The extra-terminal domain drives the role of BET proteins in transcription

The extra-terminal domain drives the role of BET proteins in transcription | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The extra-terminal domain drives the role of BET proteins in transcription Iwona Pasionek , View ORCID Profile John B. Ridenour , Agnieszka Machowska , Magdalena Donczew , Michael T. Kinter , Kevin A. Boyd , Rafal Donczew doi: https://doi.org/10.1101/2025.05.12.653540 Iwona Pasionek 1 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK, 73104, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site John B. Ridenour 1 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK, 73104, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for John B. Ridenour Agnieszka Machowska 1 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK, 73104, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Magdalena Donczew 1 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK, 73104, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael T. Kinter 2 Aging and Metabolism Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK, 73104, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kevin A. Boyd 1 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK, 73104, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rafal Donczew 1 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK, 73104, USA 3 Department of Cell Biology, University of Oklahoma Health Sciences Center , Oklahoma City, OK, 73104, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: rafal-donczew{at}omrf.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract BET proteins facilitate the transcription of most eukaryotic genes, yet the specific mechanisms underlying their function remain incompletely understood. As chromatin readers, BET proteins use tandem bromodomains to recognize acetylated lysine residues on histones and other protein partners. However, recent evidence indicates that bromodomain activity alone does not account for the full spectrum of BET protein functions, underscoring the importance of additional conserved domains. Here, we systematically evaluated all conserved domains of BET proteins and identified the extra-terminal (ET) domain as essential for cell viability, genome-wide transcription, and BET chromatin occupancy. Moreover, we demonstrate that the ET domain exerts these effects by acting as a central hub for interactions with multiple transcriptional regulators. Our findings advance the current understanding of BET protein biology and reveal potential mechanisms by which cells can evade bromodomain inhibition under pathological conditions. Introduction Bromodomains (BDs) are reader modules that recognize acetylated lysine residues on histones and other protein targets 1 . Members of the bromodomain and extra-terminal domain (BET) family are conserved chromatin readers that play central roles in transcription regulation 2 – 5 . Consistent with this function, BET proteins have been implicated in sustaining pathological transcriptional programs across various human diseases 6 , 7 . Pharmacological inhibition of BET bromodomains represents a promising therapeutic strategy for cancer and other disorders; however, resistance to BD inhibitors frequently emerges, highlighting the need for alternative approaches to target BET proteins 8 – 12 . However, a comprehensive model describing how BET proteins regulate transcription remains elusive. All three mammalian BET proteins expressed in somatic tissues (BRD2, BRD3, and BRD4) contribute to gene transcription, but BRD4 functions as the dominant BET factor, with primary roles in transcription elongation 3 – 5 , 13 , 14 . Budding yeast encode two BET proteins (Bdf1 and Bdf2, referred to here as Bdf1/2), at least one of which is required for viability. Bdf1/2 make overlapping contributions to transcription; however, similar to BRD4 in mammalian cells, yeast cells rely predominantly on Bdf1 2 , 15 . Mechanistically, Bdf1/2 support both the initiation and elongation phases of transcription 2 . Notably, mammalian BET proteins have also been implicated in transcription initiation, indicating that BET-dependent transcriptional functions are broadly conserved across eukaryotic lineages 4 , 14 , 16 . According to the canonical model, the tandem BDs serve as the primary module supporting BET protein function 1 , 8 , 17 . The multiply acetylated N-terminal tail of histone H4 is a preferred target of most BET BDs, including both BDs of Bdf1 14 , 18 , 19 . However, we previously showed that rapid depletion of H4 acetylation results in only an ∼50% reduction in Bdf1 chromatin occupancy 2 . Consistent with this observation, yeast cells remain viable in the absence of acetylated residues on the H4 tail 20 ; Bdf1 is recruited to immobilized promoter DNA even in the absence of nucleosomes 15 , 21 ; a Bdf1 mutant with inactivated BDs suppresses the temperature sensitivity caused by BDF1 deletion 18 ; and chemical inhibition of BET BDs in mammalian cells neither fully displaces BET proteins from chromatin nor abolishes their function 4 , 5 , 16 , 22 . Together, these findings indicate that BET-mediated transcriptional regulation involves additional, yet poorly understood mechanisms that do not depend on BD activity. BET proteins exhibit a modular architecture composed of conserved structured regions (BDs, motif B, and the extra-terminal (ET) domain) and conserved unstructured regions [the N- and C-terminal phosphorylation sites (NPS and CPS) and the basic interaction domain (BID)], interspersed with additional unstructured regions lacking sequence conservation ( Fig. 1A ). Although the functions of non-BD domains have not been comprehensively examined, existing evidence suggests that they play important roles. For instance, the ET domain of mammalian BET proteins serves as an interaction hub for multiple protein partners 17 , 23 – 25 , and these interactions have been proposed to contribute to BRD4-mediated transcriptional regulation, BRD4 chromatin recruitment, and the survival of certain cancer cell lines 22 , 23 , 26 – 29 . However, the in vivo significance of the ET domain for BET protein function remains unresolved. In addition, phosphorylation within the NPS and CPS regions has been implicated in enhancing BET association with chromatin and protein partners 10 , 11 , 30 . Finally, motif B and the BID have been biochemically shown to promote BET protein dimerization 31 . Download figure Open in new tab Fig. 1. The BET protein ET domain is essential for viability. ( A ) Conserved domain organization of BET proteins. BD – bromodomain, A/B – motifs A/B, BID – basic interaction domain, ET – extra-terminal domain, NPS/CPS – N/C-terminal phosphorylation sites, CTM – C-terminal motif (BRD4 long isoform only). ( B ) BDF1 variants constructed in this work. Ability to support growth in the absence of endogenous BDF1/2 is indicated. ( C ) Sequence alignment of the ET domain of BRD4 (top) and Bdf1 (bottom). Four conserved acidic residues important for interactions between BRD4 and protein partners are marked. Bdf1 coordinates are shown below the arrows. ( D ) Schematic representation of the experimental strategy for viability and genomic (SLAM-seq and ChEC-seq) assays. Experiments were performed in strains in which endogenous Bdf1/2 carried auxin-inducible degron (AID) tags and unmodified BDF1 or BDF1 variants were expressed from the TRP1 locus. Endogenous Bdf1/2 were depleted by the addition of 3-indoleacetic acid (IAA). ( E-F ) Growth assays comparing the fitness of a control strain expressing unmodified BDF1 (WT) to strains expressing BDF1 variants after 15 h exposure to IAA (long-term loss of endogenous Bdf1/2). Black markers indicate mean generation time. Error bars represent 95% confidence intervals ( n = 3). Here, we delineated the contributions of the conserved domains of BET proteins to cell viability, transcription, BET chromatin association, and the recruitment and/or maintenance of partner proteins. Our key findings establish the ET domain as essential for BET protein function in living cells and enable us to propose a revised model of BET-mediated transcriptional regulation. Results The BET protein ET domain is essential for cell survival Prior yeast genetic studies established that the BDs and NPS/CPS regions of BET proteins contribute to cell viability, but the functional significance of the remaining conserved domains has not been defined 18 , 32 . We previously demonstrated that although Bdf1 and Bdf2 have redundant roles in transcription, Bdf1 functions as the dominant BET protein in yeast 2 . Accordingly, we selected Bdf1 as our model BET factor. We generated a comprehensive collection of BDF1 variants containing deletions or targeted mutations across all conserved BET domains. When previous work suggested functional overlap, we included combinatorial perturbations affecting more than one domain. Because intrinsically disordered regions (IDRs) have been implicated in BRD4 function in mammalian cells, we also constructed a BDF1 variant with truncations in IDRs that do not overlap with the conserved unstructured NPS, CPS, or BID regions 33 . For the BDs and NPS/CPS regions, we introduced targeted mutations in residues required for BD function or in serine residues with documented phosphorylation, respectively 18 , 32 . As a comparison, we also generated a phosphomimetic BDF1 variant in which all phosphorylated serines were substituted with glutamate. In total, we constructed eighteen BDF1 variants ( Fig. 1B ). To facilitate stable expression in yeast cells, we cloned BDF1 variants or unmodified BDF1 into a minichromosomal plasmid pRS315 under the control of endogenous BDF1 promoter and 3’ untranslated region ( Table S1 ). We performed a complementation assay in a strain dependent solely on BDF1 expressed from a minichromosomal plasmid, as endogenous BDF1/2 were deleted. To test whether each BDF1 variant could support viability, we attempted to replace the plasmid carrying unmodified BDF1 and the URA3 marker with plasmids encoding the BDF1 variants, selecting for viable transformants on medium containing 5-fluoroorotic acid (5-FOA), which counterselects URA3-containing cells. Five BDF1 variants failed to support viability: the double BD-deletion and BD-mutation variants, the variants lacking both NPS and CPS regions or carrying serine-to-alanine substitutions across both regions, and the variant lacking the ET domain ( Fig. 1B ). Proper expression of these BDF1 variants was validated in experiments described below. In summary, although prior studies established in vivo requirements for the BDs and NPS/CPS regions of BET proteins in yeast 18 , 32 , our findings reveal for the first time that the ET domain is essential for cell viability. A conserved stretch of acidic residues in the ET domain is required for its function A conserved cluster of acidic residues located between the second and third helices of the ET domain has been shown to mediate BRD4 interactions with several protein partners 23 – 25 . To assess the in vivo significance of this region in Bdf1, we generated six additional BDF1 variants in which different numbers of four conserved acidic residues (amino acids 567, 569, 571, and 573) were substituted with alanine ( Figs. 1C and S1A ). Using the complementation assay described above, we found that mutation of three or more of these acidic residues is lethal in yeast, demonstrating that this region of the ET domain is essential for its function. For subsequent experiments, we selected a representative set of ET-domain mutants consisting of a single substitution (D571A; mET(1)), a double substitution (D571A, D573A; mET(2)), and a quadruple substitution (mET(4)) ( Figs. 1C and S1A ). To support subsequent experiments, we integrated all BDF1 variants, along with an unmodified BDF1 control, into the TRP1 locus of a strain in which endogenous Bdf1/2 carry auxin-inducible degron (AID) tags (RDY73; Bdf1/2-AID strain) ( Figs. 1D , and Tables S1 and S2 ) 2 . In this system, each BDF1 variant is constitutively expressed from an ectopic locus under the control of the endogenous BDF1 promoter and 3′ untranslated region, while endogenous Bdf1/2 can be rapidly depleted upon addition of 3-indoleacetic acid (IAA) 2 . Importantly, integration of an additional unmodified BDF1 copy did not affect cell viability, and the BDF1 variants were expressed at endogenous or modestly elevated levels, both in the presence and absence of endogenous Bdf1/2 ( Fig. S1B-D ). We did not detect expression of variants containing the BD2 deletion (ΔBD2, ΔBD1/2) or the deletion of the N-terminal unstructured region (ΔIDRs), suggesting that the antibody used recognizes epitopes located within these regions of Bdf1. Nevertheless, expression of the ΔBD2, ΔBD1/2, and ΔIDRs variants is supported by normal growth rates (ΔBD2 and ΔIDRs variants) and by the modest transcriptional changes observed in cells that rely on these BDF1 variants, as described below. The complementation assay identified the BET protein domains required for viability. To extend these findings, we quantified growth-rate defects associated with each BDF1 mutation. Yeast cells were cultivated in the presence of IAA, forcing dependence on the Bdf1 variants as endogenous Bdf1/2 were depleted, and generation times were calculated after ∼15 hours of growth ( Fig. 1D ). First, strains harboring BDF1 variants that failed to support viability in the complementation assay displayed complete growth inhibition, validating our earlier results. Second, whereas a single point mutation in the ET domain was well tolerated, mutation of only two conserved acidic residues caused a severe growth defect. By contrast, deletion or mutation of a single BD did not impair growth ( Fig. 1E ). Third, we found that CPS, but not NPS, deletion or mutation modestly slowed growth. Notably, the NPS/CPS phosphomimetic Bdf1 variant fully supported viability, suggesting that BET proteins function optimally in a fully phosphorylated state and that their phosphorylation may not be dynamically regulated under normal growth conditions ( Fig. 1F ). Fourth, combined loss of the B/BID regions substantially impaired growth, consistent with their proposed role in promoting BET dimerization 31 . Finally, truncation of the Bdf1 IDRs did not affect viability, indicating that the large, unstructured N-terminal region of BET proteins does not play a major biological role ( Fig. 1F ). As an additional validation, we observed similar growth rates across all viable strains relying solely on BDF1 variants expressed from minichromosomal plasmids ( Fig. S1E and Table S2 ). Collectively, these assays define the contributions of all conserved BET protein domains to yeast viability and identify a critical functional region within the ET domain. The ET domain is essential for genome-wide transcription The essential function of BET proteins is to sustain global transcriptional programs 2 – 5 . To define the contribution of each conserved BET domain to genome-wide transcription, we analyzed our collection of strains expressing BDF1 variants in the background of endogenous Bdf1/2 fused to AID tags ( Fig. 1D and Table S2 ). We previously confirmed that introducing an additional unmodified copy of BDF1 does not compromise viability and that Bdf1 protein levels are comparable when expressed from an ectopic versus endogenous locus ( Fig. S1B-D ). We further validated that this additional BDF1 copy does not substantially alter baseline transcription and largely rescues the transcriptional defects caused by depletion of endogenous Bdf1/2 ( Figs. S2A-B ). Finally, to assess whether any BDF1 variants exert dominant-negative effects in the presence of endogenous Bdf1/2, we examined growth phenotypes and found that the two strains harboring variants with combined deletion or mutation of both NPS/CPS regions displayed pronounced slow-growth phenotypes ( Fig. S2C ). In contrast to the growth assay, where cells had to rely on mutant Bdf1 variants for several generations, here we depleted endogenous Bdf1/2 for 25 minutes, followed immediately by 4-minute labeling of newly synthesized RNA with 4-thiouracil to facilitate quantification using SLAM-seq 34 ( Fig. 1D ). This approach allowed us to track direct consequences of Bdf1 mutations, including lethal mutations, with minimized bias from indirect effects that accumulate during extended exposure to adverse genetic variants. We performed three replicate experiments for all strains with (IAA) or without (dimethyl sulfoxide (DMSO)) depletion of endogenous Bdf1/2 ( Table S3 ). Variability in RNA levels between replicates in the final dataset (see Materials and Methods) was <30–40% for most genes ( Figs. S3A-B and Table S3 ). We quantified transcriptional changes associated with perturbations in BET protein domains by comparing strains expressing BDF1 variants to the strain expressing unmodified BDF1 following depletion of endogenous Bdf1/2 (IAA treatment) ( Table S3 ). To identify dominant-negative effects, we compared strains expressing BDF1 variants without depletion of endogenous Bdf1/2 to the Bdf1/2-AID parental strain (DMSO treatment), which lacks an additional BDF1 copy ( Fig. S3C and Table S3 ). Consistent with the growth assay ( Fig. S2C ), strains expressing BDF1 variants with mutations or deletions in both NPS/CPS regions (mNPS/CPS and ΔNPS/CPS) exhibited substantial transcriptional deviations (median change > 2-fold) in the presence of endogenous Bdf1/2. We observed a similar dominant-negative phenotype for the strain expressing the CPS deletion variant (ΔCPS). Consequently, we excluded ΔCPS, mNPS/CPS and ΔNPS/CPS strains from further analysis and experiments. We aligned the transcriptional changes measured following depletion of endogenous Bdf1/2 with those observed upon loss of Bdf1/2 in the Bdf1/2-AID strain. Surprisingly, only BDF1 variants lacking the ET domain or carrying mutations in all four conserved acidic residues of the ET domain (hereafter referred to as the ET domain mutation) produced substantial transcriptional defects (median decreases of 3.6-fold and 2.6-fold, respectively). By contrast, deletion or mutation of both BDs resulted in relatively modest transcriptional changes (median decreases of 1.5-fold and 1.4-fold, respectively) ( Figs. 2A and S4A ). Notably, the transcriptional defects caused by loss of ET domain function were comparable to those resulting from loss of Bdf1/2 in the Bdf1/2-AID strain (median decrease of 3.7-fold). Analysis of transcriptional changes at the level of individual genes and across previously defined major gene classes further confirmed that loss of ET domain function closely phenocopies the loss of BET proteins 35 ( Figs. 2B-C , S4B and Table S3) . Perturbations in other domains produced only minor effects on genome-wide transcription, with the exception of the CPS mutation and the combined deletion of the B/BID regions, both of which caused moderate decreases (median decreases of 1.7-fold and 1.6-fold, respectively) ( Fig. S4A ). Download figure Open in new tab Fig. 2. The ET domain is required for transcription of most genes. ( A ) Log2 changes in transcription due to selected BDF1 mutations measured by SLAM-seq after depleting endogenous Bdf1/2 for 25 min ( n = 4,836). Results of depletion of Bdf1/2 in the absence of additional copy of BDF1 are shown in the first sample from the left. Results for additional BDF1 variants are shown in Fig. S4A . ( B ) Heatmap representation of selected results from ( A ). Genes are divided into previously defined major yeast gene classes (CR – coactivator-redundant, TFIID – TFIID-dependent) 35 ( n = 4,560). ( C ) Comparison of selected results from ( A ). Pearson correlation coefficient (r) is shown ( n = 4,836). ( D ) Log2 changes in transcription due to BDF1 mutations after depleting endogenous Bdf1/2 for 3 h ( n = 4,457). Results of depletion of Bdf1/2 in the absence of additional copy of BDF1 are shown in the first sample from the left. ( E ) Volcano plot comparing the ability of the ET domain to rescue transcriptional changes following Bdf1/2 loss (log2 fold change over control strain) with associated p -values ( n = 4,715). Thresholds used: fold change – 1.5-fold; p -value – 0.05 (Welch’s t-test). ( F ) Comparison of ET domain-mediated rescue of transcription following Bdf1/2 loss (y-axis, log2 fold change over control strain) with gene dependence on Bdf1/2 (x-axis, log2 scale). Pearson correlation coefficient (r) is shown ( n = 4,715). ( G ) Growth assay comparing the fitness of the Bdf1/2-AID strain and the ET domain overexpressing strain during endogenous Bdf1/2 depletion. Samples were taken 1.5 and 3 h after DMSO or IAA addition, and growth delay was calculated by comparing generation times of IAA-treated and DMSO-treated cells. Black markers indicate mean growth delay. Error bars represent 95% confidence interval ( n = 5). Results of a Welch’s t-test are shown (** – p -value < 0.01). A modest change in transcription following BD inactivation was unexpected given the lethal phenotype associated with this mutation. We observed a similar discrepancy for deletion of the B/BID regions and mutation of two conserved acidic residues in the ET domain, both of which produced severe growth defects yet only minor transcriptional changes. To validate these findings, we extended the depletion period of endogenous Bdf1/2 to 3 hours to assess the accumulation of indirect effects on transcription. After 3 hours of IAA treatment, the Bdf1/2-AID strain exhibited a near-complete loss of genome-wide transcription (median decrease of 51-fold) ( Fig. 2D and Table S3 ). In contrast, BD1/2 mutation, ET domain double mutation, and B/BID deletion resulted in more modest median decreases of 2.9-fold, 2.5-fold, and 2.3-fold, respectively. Compared to the rapid depletion experiment ( Figs. 2A and S4A ), these results indicate that the viability defects associated with BD1/2 mutation, the ET domain double mutation, or B/BID deletion arise primarily from indirect transcriptional dysfunction. Nevertheless, the indirect effects observed after 3 hours of IAA treatment were substantially stronger for the ET domain quadruple mutation (median decrease of 22.3-fold), consistent with trends observed following 25-minute depletion of endogenous Bdf1/2. In comparison, mutation of individual bromodomains did not appreciably affect transcription after prolonged IAA treatment, consistent with the minimal viability defects associated with these BDF1 variants. Based on our findings, we hypothesized that the ET domain can at least partially compensate for the loss of BET proteins. To test this hypothesis, we overexpressed the ET domain in the Bdf1/2-AID strain ( Fig. S4C ) and quantified transcriptional changes following 25 minutes of induced Bdf1/2 depletion. First, we observed that very few genes exhibited substantial transcriptional changes as a result of ET domain overexpression in the presence of endogenous Bdf1/2 ( Fig. S4D-E and Table S3 ). Next, we compared transcriptional responses to Bdf1/2 loss between the ET domain overexpressing strain and the parental Bdf1/2-AID strain. We found that the ET domain compensated for the loss of BET proteins at many genes, with 455 genes showing statistically significant transcriptional rescue ( Fig. 2E and Table S3 ). Notably, this effect was most pronounced at strongly Bdf1/2-dependent genes ( Fig. 2F ). Consistent with these observations, the ET domain partially rescued the growth phenotype associated with BET protein loss, although prolonged exposure to IAA ultimately remained lethal ( Fig. 2G ). In summary, these results reveal a surprisingly small direct contribution of the BDs to transcription and demonstrate that the ET domain is a dominant BET protein domain in transcriptional regulation. The ET domain facilitates BET protein recruitment to chromatin To modulate transcription, BET proteins first need to be recruited to gene regulatory elements. Experiments in both yeast and mammalian cells have shown that BD function does not fully account for BET protein chromatin occupancy 2 , 4 , 15 , 16 , 22 . To identify BET protein domains that contribute to Bdf1 recruitment to chromatin, we modified selected yeast strains used in the SLAM-seq experiments by inserting a micrococcal nuclease (MNase) coding sequence before the stop codon of BDF1 variants to facilitate ChEC-seq analysis ( Tables S1 and S2 ) 36 . We previously found that ChEC-seq complements and outperforms ChIP-seq for mapping BET protein chromatin occupancy in yeast 2 . First, we validated that ectopically expressed Bdf1-MNase supports normal growth in the presence of AID-tagged endogenous BDF1 /2 ( Fig. S5A ). We also confirmed that ectopically expressed Bdf1-MNase displays a chromatin-binding pattern highly similar to that of Bdf1-MNase expressed from the endogenous locus, both in the presence and absence of endogenous Bdf1/2 ( Fig. S5B ). As in the SLAM-seq assays, sample collection was preceded by a 25-minute depletion of endogenous Bdf1/2 to minimize confounding effects from endogenous BET proteins. All experiments were performed in three biological replicates, and the replicate datasets were highly consistent ( Fig. S5C ). To evaluate the impact of targeted mutations in BET protein domains on Bdf1 recruitment, we compared data collected from strains expressing BDF1 variants to that from the control strain expressing unmodified BDF1 in the context of endogenous Bdf1/2 depletion (IAA treatment) ( Table S4 ). Consistent with the major impact of the ET domain on transcription, we found that this domain broadly contributed to Bdf1 chromatin occupancy ( Figs. 3A-C ). Surprisingly, at most gene promoters, the contribution of the ET domain to Bdf1 occupancy exceeded that of the BDs. Notably, deletion of the BDs or the ET domain produced very similar effects ( Figs. 3D and S6A-C ). Among other domains, perturbations in the CPS domain and the B/BID regions also affected Bdf1 recruitment, indicating that BET protein phosphorylation and dimerization both contribute to establishing genome-wide BET occupancy ( Fig. S6D-F ). None of the examined mutations caused substantial redistribution of Bdf1 to new loci. Next, we compared changes in Bdf1 promoter recruitment resulting from mutation of the BDs or the ET domain with levels of histone H4 lysine 12 acetylation (H4K12ac), a preferred target of Bdf1 BDs 19 . As expected, changes in Bdf1 recruitment due to BD mutation showed a modest correlation with H4K12ac levels, consistent with prior findings that BD inhibitors preferentially evict BET proteins from loci with high H4 acetylation, such as super enhancers 4 . In contrast, the effects of the ET domain on Bdf1 occupancy were independent of promoter acetylation levels ( Fig. 3E ). Finally, we observed only a weak correlation between changes in Bdf1 recruitment and transcriptional output ( Fig. S6G ), consistent with the previously reported lack of correlation between baseline Bdf1 promoter occupancy and transcription 2 . In summary, we revealed that the ET domain, not the BDs, is the primary contributor to the BET protein chromatin occupancy. Download figure Open in new tab Fig. 3. The ET domain has a major role in facilitating BET protein recruitment to chromatin. ( A ) Genome browser snapshot showing the binding patterns of unmodified Bdf1 (WT) and Bdf1 variants with mutations in the BDs or the ET domain as determined by ChEC-seq. Data for the first 100,000 bases of chromosome III are shown. ( B ) Overlap of Bdf1-bound promoters for the indicated experiments. ( C ) Occupancy of unmodified Bdf1 and Bdf1 variants with mutations in the BDs or the ET domain around the transcription start sites (TSS) of 3,388 genes whose promoters are bound by unmodified Bdf1. Genes are sorted by the TSS signal (+/-200 bp) in the WT experiment. ( D ) Comparison of log2 changes in Bdf1 occupancy due to mutation or deletion of the BDs or the ET domain. Bdf1 occupancy was calculated as the sum of signal within the (-100, 100) bp window around the Bdf1 promoter peak summit. Pearson correlation coefficient (r) is shown ( n = 3,388). ( E ) Comparison of H4K12ac level at promoters 2 and log2 changes in Bdf1 occupancy due to mutations in the BDs or the ET domain. Pearson correlation coefficient (r) is shown. The ET domain supports TFIID recruitment to gene promoters Both yeast and mammalian BET proteins have been linked to the coactivator TFIID, which regulates transcription initiation 2 , 14 , 15 . Our prior work demonstrated that Bdf1/2 are required for transcription initiation, and that loss of Bdf1/2 modestly decreases TFIID promoter occupancy 2 . Building on this background and our new findings, we hypothesized that a major function of the ET domain in transcription regulation is to facilitate TFIID recruitment. To test this hypothesis, we applied ChEC-seq to quantify changes in TFIID occupancy in cells relying solely on Bdf1 variants after rapid depletion of endogenous Bdf1/2. We inserted an MNase coding sequence into the TFIID subunit Taf1 in three strains previously used in the SLAM-seq assays: the control strain carrying an unmodified additional copy of BDF1 , and strains carrying BDF1 variants with mutations in the ET domain or the BDs (mET(4) and mBD1/2, respectively). After depleting endogenous Bdf1/2, we quantified changes in Taf1 recruitment by comparing strains expressing BDF1 variants to the control strain. Experiments were performed in three biological replicates, and the replicate datasets were highly reproducible ( Fig. S7A ). We found that both the ET domain and the BDs contributed to TFIID recruitment to promoters; however, the ET domain contributed more prominently at most loci ( Fig. 4A-B ). Next, we compared changes in TFIID occupancy resulting from Bdf1 mutations with changes in transcription and observed only a modest correlation ( Fig. S7B ). Consistent with this finding, we previously reported that transcription is uncoupled from baseline TFIID promoter occupancy, and that changes in TFIID occupancy due to complete loss of BET proteins do not correlate with transcriptional output 2 , 35 . In summary, our results demonstrate that the ET domain plays a major role in TFIID recruitment to gene promoters. Download figure Open in new tab Fig. 4. The ET domain facilitates recruitment of TFIID to promoters. ( A ) Genome browser snapshot showing Taf1 (TFIID) binding pattern in the context of unmodified Bdf1 or Bdf1 variants with mutations in the BDs or the ET domain as determined by ChEC-seq. Data for the first 100,000 bases of chromosome III are shown. ( B ) Taf1 occupancy around TSS of 3,103 genes whose promoters are bound by Taf1 2 , in the context of unmodified Bdf1 or Bdf1 variants with mutations in the BDs or the ET domain. Genes are sorted by the signal around TSS (+/-200 bp) in the WT experiment. The ET domain engages its targets using a conserved mechanism Results described thus far show that the ET domain is a primary functional module of Bdf1 and that its impact on transcription is at least partially explained by its role in facilitating TFIID recruitment. Consistent with the latter finding, the ET domain of BRD4 has been proposed to modulate transcription by mediating interactions with protein partners 17 , 22 , 23 . However, only a limited number of interactions involving the ET domain of mammalian BET proteins have been experimentally validated, and it remains unknown how many factors associate with the ET domain or what the functional consequences of those interactions are. To begin addressing these questions, we used chromatin mass spectrometry (chromatin-MS) to identify candidate factors – beyond TFIID – that may depend on Bdf1/2 for recruitment to chromatin. We obtained reliable data for 724 of 2399 yeast nuclear proteins and found that 78 exhibited decreased chromatin association following rapid Bdf1/2 depletion. As validation of our approach, this list included two TFIID subunits. Notably, we also identified additional proteins implicated in transcription regulation, including Bur1/CDK9, Spt6, the PAF1 complex (PAF1C) subunit Rtf1, subunits of the RSC, INO80, and ISW2 chromatin remodeling complexes, and several factors involved in mRNA processing ( Figs. 5A and S8A and Table S5 ). Download figure Open in new tab Fig. 5. The ET domain of yeast and mammalian BET proteins uses the same mechanism to engage its targets. ( A ) Volcano plot comparing –log10 p -value and log2 change in abundance for chromatin-associated proteins following Bdf1/2 depletion. Data for reliably detected nuclear proteins are shown ( n = 724). ( B ) Schematic representation of the experimental strategy for identifying high-confidence ET-interacting motifs (ETMs) in S. cerevisiae nuclear proteins. ( C ) Functional domains of Taf7. Interaction with Bdf1 was previously mapped to the unstructured N-terminal region of Taf7 15 , which contains four high-confidence ETMs (detail). The TAFII55 region mediates interaction with Taf1 and is conserved across Taf7 homologs in other eukaryotes. The predicted C-terminal coiled-coil domain is poorly characterized. Other regions of Taf7 are largely unstructured. ( D ) AlphaFold2 (AF2) predicted structures for the Bdf1 ET domain in association with four putative ETMs in the Bdf1-interaction region of Taf7. Confidence metrics (ipSAE, pDockQ2, and LIS) and the number of interfacial contacts supporting structural predictions are shown. ( E ) Y2H analysis of the interaction between the Bdf1 ET domain and Taf7. Plasmids expressing the Bdf1 ET domain (WT or mut), Taf7 (WT or mut), or empty vector (EV) were co-transformed into the host strain. Cells were serially diluted, spotted on indicated media, and imaged after three days of growth ( n = 3). ET mut – ET domain with four conserved acidic residues substituted with alanine. Taf7 mut – Taf7 with basic residues in the four ETMs in the N-terminal region (first 101 amino acids 15 ) substituted with alanine. Based on proteomic and structural biology studies, the BRD4 ET domain has been proposed to recognize two consensus motifs – Φ[+]Φ[+] or (Φ/[+])ΦΦ[+](Φ/[+]), where Φ is M, L, V, I, or F and [+] is K or R – referred to here as ET-interacting motifs (ETMs) 17 , 24 , 25 . Screening the yeast nuclear proteome revealed 2418 ETMs in 1253 proteins, including 60 of the 78 proteins identified in our chromatin-MS experiment ( Fig. 5B and Table S6 ). Given this large number of putative interactors, we used AlphaFold2 to model interactions between the Bdf1 ET domain and candidate ETMs 37 . As an initial validation of this approach, we modeled the BRD4 ET domain with selected partner peptides and observed strong similarity between the predicted structures and published experimental structures, including formation of an interfacial β-sheet involving the unstructured loop between the α2 and α3 helices of the ET domain, which adopts a β-strand configuration when bound to partner peptides ( Fig. S8B-C ). Modeling the Bdf1 ET domain with the same peptides similarly produced structures consistent with experimental data, including conditional interfacial β-sheet formation, suggesting a conserved mode of interaction ( Fig. S8D ). We next folded the Bdf1 ET domain with each of the 2418 ETMs identified in yeast nuclear proteins using two implementations of AlphaFold2 and scored models using multiple confidence metrics and interfacial β-sheet establishment (see Materials and Methods) ( Table S7 ). To test whether our approach could identify bona fide ET domain interactors, we focused on the TFIID subunit Taf7, a confirmed Bdf1-interacting partner. The interaction was previously mapped to the N-terminal 100 amino acids of Taf7 15 . Within this region, we identified four putative ETMs, each ranking highly in accessibility and model confidence ( Figs. 5C and S8E-F and Table S7 ). Structural models of these four ETMs in complex with the Bdf1 ET domain were well supported and showed conditional interfacial β-sheet formation ( Figs. 5D and S9A ). Using a yeast two-hybrid system, we tested whether the Bdf1-Taf7 interaction is mediated by the ET domain and by ETMs in the Taf7 N-terminal region. We found that the Bdf1 ET domain interacts with Taf7 and that this interaction was abolished either by mutating the ET domain or by substituting basic residues with alanine in all four Taf7 ETMs ( Fig. 5E ). Based on these results, together with the observation that conditional β-sheet formation correlates with higher model confidence ( Fig. S9B-C ), we defined 770 high-confidence ETMs (AlphaFold RSA ≥ 0.24 and FoldScore = 1) present in 596 nuclear proteins ( Table S7 ). This high-confidence ETM-containing set was enriched for proteins previously associated with Bdf1 function ( Fig. S9D ). Taken together, these findings provide a mechanistic basis for Bdf1-dependent recruitment of TFIID, demonstrate that the ET domains of yeast and mammalian BET proteins engage their targets using a conserved interaction mechanism, and validate our approach for systematically identifying and characterizing interacting partners of the BET protein ET domain. Multiple interactions mediated by the ET domain contribute to gene transcription To further define the mechanistic basis by which the ET domain regulates transcription, we selected the TFIID subunit Taf7 and several additional regulatory factors for detailed analysis. Specifically, we focused on Bur1/CDK9, Spt6, the PAF1C subunit Rtf1, two conserved mRNA-processing factors (Rat1/XRN2 and Rna14/CSTF3), and Pob3, a subunit of the histone chaperone FACT. Each of these proteins contains at least one high-confidence ETM ( Fig. S10 and Table S7 ), and all except Pob3 showed decreased chromatin association following Bdf1/2 depletion in our chromatin-MS experiment ( Fig. 5A ). Notably, both FACT subunits Pob3 and Spt16 were previously identified as putative Bdf1-associated proteins by mass spectrometry 18 , and CDK9, FACT, SPT6, PAF1C, XRN2 and CSTF3 have been linked to BET proteins in mammalian cells 5 , 13 , 22 , 38 . However, the mechanistic basis of an association involving BET proteins and any of these factors except CDK9 has not been established. We first used parallel reaction monitoring mass spectrometry (PRM-MS), a highly quantitative MS method that targets predefined peptides 39 , to measure changes in chromatin association of Bur1, FACT, Spt6, PAF1C, Rat1, and Rna14 following Bdf1/2 depletion. Because Rtf1 could not be reliably detected by PRM-MS, we instead quantified changes in Paf1, the major subunit of PAF1C. For all factors tested, we observed an approximately 50% loss of chromatin association, without a corresponding decrease in total protein levels ( Fig. 6A and Table S5 ). Second, we used a yeast two-hybrid system to test interactions between predicted high-confidence ETMs in Bur1, Pob3, and Spt6 ( Fig. S10 and Table S7 ) and the ET domain of Bdf1. We found that the Bdf1 ET domain interacted with all predicted targets and that, with the exception of one Pob3 ETM, these interactions were abolished by mutation of either the ET domain or the ETMs ( Fig. S11A ). Based on these results, we generated yeast strains stably expressing variants of Taf7, Bur1, Spt6, or Pob3 in which all identified ETMs were mutated by substituting lysine or arginine residues with alanine. All ETM-mutant strains exhibited moderate growth defects, indicating the functional importance of ET-mediated interactions with the analyzed factors ( Fig. 6B ). Next, we applied SLAM-seq to determine whether ETM-mutant strains display alterations in transcriptional programs ( Table S3 ). In the case of the Taf7 and Bur1 mutant strains, we observed downregulation of large subsets of genes ( Fig. 6C ). Conversely, we detected upregulation of many genes in the the Pob3 and Spt6 mutant strains ( Fig. 6C ), suggesting that the interactions between BET proteins and FACT or Spt6 are important for reducing and/or fine-tuning the transcription of certain genes. Consistent with this interpretation, prior studies reported increased coding transcription of subsets of genes upon impaired FACT or Spt6 function 40 – 42 . Importantly, across all ETM-mutant strains, the subsets of genes that passed the significance threshold overlapped with genes that are strongly sensitive to Bdf1/2 loss ( Fig. S11B ). In summary, our findings demonstrate that ET domain–mediated interactions of BET proteins support chromatin occupancy of multiple partner proteins and enable optimal transcription of target genes. Download figure Open in new tab Fig. 6. The ET domain modulates transcription by recruiting key regulatory factors. ( A ) Abundance of selected proteins in chromatin-enriched fractions (top panel) or whole cell extract (bottom panel) following Bdf1/2 depletion as determined by parallel reaction monitoring mass spectrometry. Central lines indicate mean protein abundance. Error bars represent standard deviation of the mean ( n = 6). Results of a Welch’s t-test are shown (* – p -value < 0.05, ** – p -value < 0.01, ns – not significant). ( B ) Growth assays comparing the fitness of strains expressing variants of indicated proteins carrying targeted ETM mutations to control strains expressing a wild-type variant of the protein from the same genomic locus. Black markers indicate mean generation times. Error bars represent 95% confidence interval ( n = 5). Results of a Welch’s t-test are shown (* – p -value < 0.05, ** – p -value < 0.01, *** – p -value < 0.001). ( C ) Volcano plots showing log2 change in transcription due to mutation of ETMs in indicated proteins ( n = 4,498). The numbers of genes showing significant up- or down-regulation are indicated. Thresholds used: fold change – 1.5-fold; p -value – 0.05 (Welch’s t-test). Discussion Discovery of specific inhibitors of BET BDs brought the promise of effective treatments for cancer and other diseases. However, the clinical application of BD inhibitors has faced significant challenges due to emerging resistance, cellular heterogeneity, and an incomplete understanding of BET protein biology. In this work, we used a highly versatile yeast model system to define the contributions of all conserved BET protein domains to cell viability, gene transcription, and BET chromatin occupancy. We found that the ET domain, rather than the BDs, constitutes the primary functional module of BET proteins. These results both complement and challenge existing models of BET-mediated transcription, reconcile conflicting observations from prior studies, and suggest new directions for future research. Many studies have reported BET protein recruitment and function that persist despite BD inhibition 5 , 10 – 12 , 22 . Through genetic analysis, we identified five BET protein domains essential for cell viability, including the BDs, the ET domain, and the phosphorylated NPS/CPS regions. Although the requirement for at least one BD and for the NPS/CPS regions was previously established 18 , 32 , our work is the first to demonstrate that the ET domain is essential for cell survival. Consistent with the viability defects, we found that the ET domain makes major contributions to global transcription and to BET protein recruitment to chromatin, exceeding the contributions of the BDs. In addition, guided by prior studies 23 – 25 , we identified conserved acidic residues required for ET domain function in vivo . The ET domain has been proposed as a feasible drug target for modulating aberrant transcription in human disease 17 , 28 , 29 . Structural information for the ET domain in complex with selected peptide ligands is available, and our data show that the mechanistic basis of ET-mediated interactions is conserved between yeast and mammals. Thus, the yeast model system can serve as a valuable platform to evaluate the ability of small-molecule inhibitors to disrupt ET domain function. Importantly, relative sensitivity of cancer cells to BD inhibition or BET protein degradation differs depending on the tissue of origin and other characteristics, suggesting a possibility of tailored therapeutic approaches 4 , 8 , 10 . Altogether, our findings provide a framework and rationale for developing approaches to modulate BET protein activity by targeting the ET domain either in place of, or in combination with, BD inhibition. Other phenotypes we observed for Bdf1 mutants also complement and extend prior studies. Motif B and the BID were shown to facilitate BRD4 dimerization in biochemical assays, and deletion of motif B caused dissociation of BRD2 from chromatin 31 , 43 . We observed a significant growth phenotype correlated with changes in Bdf1 recruitment and, to a lesser extent, changes in transcription resulting from deletion of the B/BID regions. Together with prior studies, these findings indicate that BET proteins function as dimers, and future work should address how dimerization modulates BET protein recruitment and activity. For example, an important regulatory layer may arise from the relative ability of BET proteins to form homo- and heterodimers. Phosphorylation of BET proteins in the NPS and CPS regions has been linked with resistance to BD inhibition, interactions with protein partners, and the propensity of BET proteins to dimerize 10 , 11 , 30 , 31 . It was proposed that NPS/CPS phosphorylation may act as a switch that modulates BET protein dimerization and affinity for distinct partners 30 , 31 . In contrast, our finding that a phosphomimetic BDF1 variant supports normal growth and causes only mild changes in transcription and Bdf1 chromatin occupancy suggests that dynamic regulation of BET protein phosphorylation is not required for BET protein function. Moreover, prior work proposed distinct roles for the NPS and CPS regions, with NPS phosphorylation linked to BD activity and CPS phosphorylation suggested to influence ET domain function 10 , 30 . We found that mutation or deletion of NPS did not impair BET protein function, mutation or deletion of CPS was detrimental, and simultaneous perturbation of both regions was lethal. Based on these observations, we propose that phosphorylation of the NPS and CPS regions supports the same functional role or roles of BET proteins. Nevertheless, the biological significance of BET protein phosphorylation remains unresolved and warrants further investigation. Two recent studies provided mechanistic explanations for BD-independent functions of mammalian BET proteins in transcription 5 , 22 . Zheng et al. 5 demonstrated that the C-terminal fragment specific to the long isoform of BRD4 can independently promote the release of paused RNA polymerase II (Pol II) through interaction with CDK9/CCNT1 (the P-TEFb complex) and can rescue transcription of a subset of genes following depletion of endogenous BRD4. Yeast lack an equivalent of the long BRD4 isoform, and transcription in yeast does not involve a regulated Pol II pause-and-release mechanism. Our experiments demonstrated that the Bdf1 ET domain interacts with the CDK9 homolog Bur1, and that mutation of Bur1 ETMs impairs cell growth and transcription of a subset of genes. As additional validation, we previously reported defects in Pol II processivity at many of the same genes following Bdf1/2 depletion 2 . Taken together, these observations support a conserved association between BET proteins and Bur1/CDK9 in yeast and mammals, even though the mechanistic basis of the interaction differs. In yeast, the roles of BET proteins in transcription initiation and elongation are mediated primarily by a single Bdf1 isoform and likely by the same functional domain, the ET domain. In mammalian cells, evolutionary acquisition of the long BRD4 isoform may reflect the need to separate BRD4 functions in transcription elongation from other activities also mediated by the short BRD4 isoform, which may be shared with BRD2 and BRD3 14 , 17 , 23 . Why such functional separation became advantageous in the context of more complex gene regulatory networks remains an important question for future investigation. Zhang et al. 22 reported that BRD4 activates transcription at estrogen receptor binding sites through BD-independent associations with SPT5, SPT6, and PAF1C. Consistent with this finding, we observed that Bdf1/2 depletion reduces chromatin association of Spt6 and PAF1C. Spt5, Spt6, and PAF1C subunits each contain high-confidence ETMs, and mutation of Spt6 ETMs resulted in growth defects and dysregulated transcription at many Bdf1/2-dependent genes. Zhang et al. 22 also showed that association with the transcriptional coactivator Mediator contributes to BD-independent BRD4 recruitment. Importantly, the mechanistic basis of the established association between BET proteins and Mediator remains unresolved. Because several Mediator subunits are present in our high-confidence ETM list, it is plausible that BET proteins directly interact with Mediator via the ET domain. Finally, although Bdf1/2 depletion modestly reduces Mediator chromatin occupancy 2 , Bdf1 chromatin association may also be regulated by interactions with Mediator, consistent with the substantial loss of Bdf1 chromatin occupancy observed upon ET domain mutation. In summary, we provided evidence that the ET domain has a critical role in transcription by serving as a hub for interactions with multiple protein partners, including the transcription coactivator TFIID, Bur1/CDK9, and the histone chaperones Spt6 and FACT. Our analysis indicates that the repertoire of direct ET domain interactors is likely substantially larger. The experimental framework described here can be applied to identify and functionally characterize ET domain interactions with additional transcription regulators in both yeast and mammalian cells. We propose that multiple protein-protein interactions mediated by the ET domain facilitate recruitment of diverse components of the transcription machinery, as well as recruitment of BET proteins themselves. Importantly, this model is consistent with current views of transcription regulation based on the formation of transcriptional condensates through transient interactions among multivalent proteins, and with the propensity of BET proteins to promote condensate formation at gene regulatory elements 44 , 45 . Despite fundamental and clinical relevance, many aspects of the biology of BET proteins remain poorly understood. We revealed critical roles of the BET protein ET domain in sustaining global transcription and facilitating BET protein recruitment to chromatin. Our findings provide a new mechanistic explanation for the failure of BD inhibitors to abolish BET protein chromatin occupancy and function in clinical settings and suggest directions for future investigations toward a comprehensive model of the complex roles of BET proteins in transcription regulation. Limitations of the study First, the experiments described in this study were carried out under optimal growth conditions (rich medium, 30°C). As such, it remains to be determined if the contributions of distinct BET protein domains to transcription vary depending on the nutrient availability, stress, or external signaling cues. Second, although prior evidence suggests that Bdf1 is the primary BET protein in yeast cells 2 , 15 , we cannot exclude the possibility that Bdf2 plays important and non-redundant roles in transcription that are not addressed by our experiments. For example, Bdf2 may be necessary for gene activation rather than steady-state transcription, analogous to proposed roles for BRD2 and BRD3 in mammalian cells 46 . Consistent with this idea, our recent study identified the BDF2 promoter as a target of numerous sequence-specific transcription factors, the BDF2 gene was shown to be regulated by two distinct RNA degradation mechanisms in response to environmental conditions, and Bdf2 was among a small subset of yeast proteins with an unusually short half-life, suggesting dynamic regulation of BDF2 expression transcriptionally, post-transcriptionally, and post-translationally 47 – 49 . Finally, there is a possibility that small amounts of endogenous Bdf1/2 remaining after depletion using the AID system may have contributed to confounding effects in our experiments, potentially biasing our interpretation. However, we consider this unlikely, as we observed highly similar results between growth retardation experiments based on Bdf1/2 depletion and BDF1/2 gene deletion, in which growth was supported by BDF1 variants expressed from minichromosomal plasmids. Materials and Methods Yeast cell growth All Saccharomyces cerevisiae and Schizosaccharomyces pombe strains used in this study are listed in Table S2. For strain construction, S. cerevisiae strains were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose, 20 μg/ml adenine sulfate) or in synthetic complete (SC) media (0.17% yeast nitrogen base without ammonium sulfate or amino acids [BD Difco], 0.5% ammonium sulfate, 40 μg/ml adenine sulfate, 0.6 mg/ml amino acid dropout mix, supplemented with 2 μg/ml uracil and 0.01% other amino acids to complement auxotrophic markers) at 30°C. Standard amino acid dropout mix contains 2 g each of Tyr, Ser, Val, Ile, Phe, Asp, and Pro and 4 g each of Arg, Thr, Lys, and Met. 1.5% Bacto-agar (BD Difco) was added to medium as required for growth on solid media. For complementation assays, S. cerevisiae strains were grown in SC medium lacking Leu to select for cells carrying a minichromosomal plasmid with BDF1 variants and plated onto medium containing 5-fluoroorotic acid (5-FOA) to shuffle out the wild-type BDF1 plasmid carrying the URA3 marker. For genomic and growth assays, S. cerevisiae cells were grown in YPD medium at 30°C with shaking. Schizosaccharomyces pombe cells were grown in YE medium (0.5% yeast extract, 3% glucose) at 30°C with shaking. In experiments involving degron-mediated depletion of target proteins, cells were treated with 1 mM IAA dissolved in DMSO, or with DMSO alone, for 25 min or 3 h to induce protein degradation, followed by protocol-specific steps. For SLAM-seq and ChEC-seq experiments, S. cerevisiae cells were collected between an OD600 of 0.5 and 0.7, and S. pombe cells were collected at an OD600 of 1.0. For growth assays, cells were grown from an OD600 of 0.0001–0.0005 for 15 h with or without addition of IAA or DMSO. A minimum of five biological replicates were collected for growth assays using strains expressing BDF1 variants from a minichromosomal plasmid. Three biological replicates were collected for all other experiments unless otherwise indicated. Plasmid and strain construction Plasmids and S. cerevisiae strains were constructed using standard methods and are described in Tables S1 and S2 , respectively. Previously described plasmids or strains were used as indicated 36 , 50 – 52 . Proteins were chromosomally tagged by yeast transformation and homologous recombination of PCR-amplified DNA. For ChEC-seq experiments, proteins were tagged with 3xFLAG-MNase::TRP1 or 3xFLAG-MNase::HYG using pGZ110 36 or pMD75 (this work) plasmids, respectively. ET domain overexpression The gene fragment encoding the Bdf1 ET domain was cloned into the pMD1 plasmid under the control of the GPM1 promoter and the BDF1 terminator and placed in frame with the RPL25 nuclear localization signal and a 3xV5 epitope. The resulting plasmid pJR15 was integrated into the TRP1 locus of strain RDY73 (Bdf1/2-AID) to generate strain RDY388. Western blot analysis A 1 ml fraction of culture was collected and pelleted from strains after treatment with IAA or DMSO, washed with 500 μl water, and resuspended in 100 μl of yeast whole cell extract buffer (60 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.0025% bromophenol blue). After heating at 95°C for 5 min, samples were centrifuged at 21,000 × g for 5 min and analyzed by SDS-PAGE and western blot. Protein signals were visualized using the Odyssey DLx scanner (Li-Cor) and quantified using the Odyssey Empiria Studio program (Li-Cor). Fluorescent signal from total protein staining (Li-Cor Total Protein Stain workflow) was used to normalize signals for target proteins. SLAM-seq SLAM-seq was performed as previously described 34 . Briefly, following DMSO or IAA treatment, S. cerevisiae cultures were treated with 5 mM 4-thiouracil (4tU) in DMSO or with DMSO alone for 4 min. Cells were immediately fixed in cold methanol on dry ice and stored at –80°C. The OD600 at the time of collection was ∼0.7. S. pombe cultures were treated with 4tU and collected using the same procedure. Three biological replicates were collected for all experiments. RNA purification and DNase I treatment were performed using the Quick-RNA Fungal/Bacterial Miniprep kit (Zymo Research) according to the manufacturer’s recommendations under reducing conditions, as described. Five micrograms of total RNA was used for alkylation. A total of 200 ng of alkylated RNA was used to construct 3′ mRNA sequencing libraries. Libraries were sequenced on a NovaSeq 6000 (Illumina) at the Oklahoma Medical Research Foundation (OMRF) Clinical Genomics Center (Oklahoma City, OK, USA). SLAM-seq data analysis Data (paired-end 150 bp reads) were analyzed as previously described 34 . Preprocessing and processing steps were implemented as a Snakemake workflow ( https://github.com/DonczewLab/SLAM-Seq_Analysis ). Briefly, reads were preprocessed using fastp (version 0.23.2) 53 and bbduk (BBMap version 39.06) ( https://sourceforge.net/projects/bbmap/ ). Reads were then aligned and processed using SLAM-DUNK (version 0.4.3) 54 . Total read counts were defined as the number of reads remaining after alignment and filtering in SLAM-DUNK. T>C read counts were defined as the number of reads containing ≥2 T>C conversions. Genes with zero T>C read counts in one or more replicate samples among all 25 min depletion experiments were excluded. Normalization and differential expression analyses were performed on T>C read counts using DESeq2 (version 1.38.3) 55 . Normalization factors were calculated using total read counts for 25 min depletion experiments. Because large changes in total read counts were expected after 3 h depletion, spike-in read counts were used for normalization in those experiments. Genes that did not meet DESeq2 criteria for fold-change and/or p -value calculation were excluded from downstream analyses. ChEC-seq ChEC-seq was performed as previously described with minor modifications 2 , 56 , 57 . Briefly, 50 ml S. cerevisiae cultures were pelleted at 2000 × g for 3 min. Cells were resuspended in 1 ml of Buffer A (15 mM Tris-HCl [pH 7.5], 80 mM KCl, 0.1 mM EGTA, 0.2 mM spermine [Millipore Sigma], 0.3 mM spermidine [Millipore Sigma], 1X protease inhibitors [2 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 2 mM PMSF]), transferred to a 1.5 ml microcentrifuge tube, and pelleted at 1500 × g for 30 s. Cells were washed twice with 1 ml of Buffer A and resuspended in 570 μl of Buffer A. Thirty microliters of 2% digitonin (Millipore Sigma) was added to a final concentration of 0.1%, and cells were permeabilized for 5 min at 30°C with shaking (900 rpm). Samples were incubated with 0.2 mM CaCl2 for 5 min at 30°C with shaking. A 100 μl fraction of the suspension was mixed with 100 μl of Stop Solution (400 mM NaCl, 20 mM EDTA, 4 mM EGTA). Samples were then incubated with 0.4 mg/ml Proteinase K (Thermo Fisher Scientific) for 30 min at 55°C. DNA was purified using the ChIP DNA Clean and Concentrator kit (Zymo Research) and eluted in 30 μl of elution buffer. Samples were treated with 0.3 mg/ml RNase A (Thermo Fisher Scientific) for 20 min at 37°C. Thirty microliters of Sera-Mag Speedbeads (Cytiva), prepared as described (dx.doi.org/10.17504/protocols.io.x54v9p7b1g3e/v2), were added, and samples were incubated for 10 min at room temperature. Supernatants were transferred to a new tube, and the volume was adjusted to 175 μl (10 mM Tris-HCl, pH 8.0). DNA was purified again using the ChIP DNA Clean and Concentrator kit and eluted in 30 μl of Elution Buffer. Libraries were prepared using the Next Ultra II DNA Library Prep kit (New England Biolabs) as described 58 and sequenced on a NovaSeq 6000 at the OMRF Clinical Genomics Center. ChEC-seq data analysis Data (paired-end 150 bp reads) were analyzed as previously described with minor modifications 2 , 56 . Initial preprocessing and processing steps were implemented as a Snakemake workflow ( https://github.com/DonczewLab/ChEC-Seq_Analysis ). Briefly, reads were preprocessed using bbduk (BBMap version 39.06) ( https://sourceforge.net/projects/bbmap/ ) and aligned using Bowtie 2 (version 2.5.0) 59 . BAM files were generated, sorted, and indexed using Samtools (version 1.18) 60 . Genome coverage was calculated and normalized by counts per million (CPM) using deepTools2 (version 3.5.4) 61 . Average genome coverage across replicates was calculated using BEDTools (version 2.30.0) 62 and bedGraphToBigWig 63 . SAM files for S. cerevisiae were converted to tag directories using the HOMER 64 ‘makeTagDirectory’ tool. Peaks were called using the HOMER ‘findPeaks’ tool with optional arguments set to ‘-o auto -C 0 L 2 F 2’, using the free MNase dataset as a control 2 . These settings apply a default false discovery rate (FDR; 0.1%) and require peaks to be enriched twofold over the control and twofold over the local background. Resulting peak files were converted to BED format using the ‘pos2bed.pl’ program. For each peak, the summit was defined as the midpoint between the peak borders. For promoter assignment, the list of annotated ORF sequences (excluding entries classified as ‘dubious’ or ‘pseudogene’) was downloaded from the SGD website ( https://www.yeastgenome.org ). Data for 5888 genes were merged with TSS positions 65 . If TSS annotation was missing, the TSS was manually assigned at −100 bp relative to the start codon. At this stage, biological replicates were averaged for each sample. Peaks were assigned to promoters if their summit was located between −300 and +100 bp relative to the TSS. When more than one peak mapped to a promoter, the peak closest to the TSS was used. Promoters bound in at least two out of four replicate experiments were included in the final list. To quantify log2 changes in promoter occupancy, signal per promoter was calculated as the sum of normalized reads within a 200 bp window centered on the promoter peak summit. ET-interacting motif discovery Putative ET-interacting motifs (ETMs) were identified using SLiMSearch (version 4) 66 . The previously described ETMs Φ[+]Φ[+] and (Φ/[+])ΦΦ[+](Φ/[+]), where Φ is M, L, V, I, or F and [+] is K or R 17 , were used as input to query the S. cerevisiae proteome. ETMs were limited to those present in nuclear proteins based on evidence in AllianceMine 67 . Overlapping and/or adjacent ETMs were collapsed. Disorder propensity and accessibility were calculated in SLiMSearch using IUPred2A 68 and AlphaFold RSA 69 , respectively. Structural predictions Initial folding experiments involving characterized BRD4-interacting peptides and the BRD4 ET domain or the Bdf1 ET domain were performed using a locally installed instance of AlphaFold2 (version 2.3.2) 37 , 70 . Experimental structures were obtained from the Protein Data Bank (PDB) 71 . ChimeraX (version 1.10.1) 72 was used to visualize structures and calculate structural similarity and interfacial contacts. Similarity between experimental and predicted structures was calculated as root mean square deviation (RMSD). Contacts were calculated as the number of interfacial residue pairs ≤ 4.0 Å apart with a maximum predicted aligned error (PAE) ≤ 5.0 Å. ETMs in nuclear proteins ( n = 2418) were folded with the Bdf1 ET domain using two implementations of AlphaFold2 : the locally installed AlphaFold2 instance (as above) and ColabFold Batch (version 1.5.5) 73 run on Google Colaboratory. Eight amino acids N-terminal and eight amino acids C-terminal to each ETM were included in folding experiments. Confidence metrics were calculated using the ipSAE program (version 3) 74 . Cutoffs of PAE ≤ 10 Å and distance ≤ 10 Å were used to compute ipSAE scores. Combined ipSAE confidence was calculated as the unweighted z-score average (scaled 0-1) across ipSAE scores of the top-ranked AlphaFold2 and ColabFold models. Combined pDockQ2 confidence was calculated as the unweighted z-score average (scaled 0-1) across pDockQ2 scores of the top-ranked AlphaFold2 and ColabFold models. The DSSP program (version 4.5.6) 75 was used to assign secondary structure elements (i.e., interfacial β-sheet establishment) in predicted structures. FoldScore was calculated based on conditional interfacial β-sheet establishment across top-ranked AlphaFold2 and ColabFold models: a FoldScore of 1 indicates that interfacial β-sheets with the same orientation were assigned in both models; a FoldScore of 0.5 indicates that interfacial β-sheets with different orientations were assigned in the two models or that an interfacial β-sheet was assigned in only one model; and a FoldScore of 0 indicates that an interfacial β-sheet was not assigned in either model. Chromatin mass spectrometry Chromatin-enriched fractions and whole cell extract were collected using chromatin-enriched fractionation (ChEF) as previously described, with modifications 76 . Briefly, S. cerevisiae was grown in 200 ml YPD medium at 30°C with shaking at 220 rpm to an OD600 of ∼0.5 and split into two 100 ml cultures. Cultures were treated with IAA or DMSO for 25 min. Cells were pelleted at 2000 × g for 3 min, washed once with sterile water, and stored at –80°C. The OD600 at the time of collection was ∼0.7. A minimum of five biological replicates were collected. Cells were resuspended in 1 ml ChEF buffer 1 76 supplemented with 1X phosphatase inhibitors (Apex Biotechnology), 1X deacetylase inhibitors (Apex Biotechnology), and 1X protease inhibitors (see above). Cells were combined with 1.5 ml disruption beads and processed in a Mini-Beadbeater-24 (Biospec Products) at 3800 rpm for 30 s followed by incubation on ice for 2 min, repeated 10 times. Cell lysate was recovered as previously described 77 and cleared twice by centrifugation at 500 × g for 5 min at 4°C. A fraction of the cleared lysate represented whole cell extract. The remaining lysate was processed as described previously 76 , except that ChEF buffer 2 was supplemented with 1X phosphatase inhibitors, 1X deacetylase inhibitors, and 1X protease inhibitors. Chromatin-enriched fractions were resuspended in 50 mM Tris-HCl (pH 7.5). A total of 15 pmoles of bovine serum albumin (BSA) was added to 100 μg of total protein from chromatin-enriched fractions or 200 μg of total protein from whole cell extracts as an internal standard. Samples were processed as previously described 78 . Samples from chromatin-enriched fractions were analyzed using a Q Exactive Plus mass spectrometer system (ThermoFisher Scientific) in data-independent acquisition (DIA) mode with a 20 m/z window. Samples from whole cell extracts were analyzed using an Exploris 480 mass spectrometer system (ThermoFisher Scientific) in DIA mode with a 10 m/z window. Data were analyzed using DIA-NN (version 1.9.2) 79 searching against the S. cerevisiae UniProt protein database (downloaded from ENA/EMBL, accession GCA_000146045.2). Parallel reaction monitoring mass spectrometry Whole cell extracts and chromatin-enriched fractions were collected as described above. A total of 14 pmoles of BSA was added to 60 μg of total protein from chromatin-enriched fractions or 100 μg of total protein from whole cell extracts as an internal standard. Samples were processed as previously described 78 and analyzed using a Stellar mass spectrometer system (ThermoFisher Scientific) in PRM mode with a resolution of 1.2 Da and a scan rate of 67 kDa/sec. Data were processed using Skyline (version 25.1) 80 to identify and integrate chromatographic peaks for a defined set of validated peptides. Retention times were calibrated using BSA and trypsin peptides. Protein abundance was calculated as the geometric mean of a minimum of two monitored peptides normalized to the BSA internal standard and expressed as pmol per 10 µg of total protein. Yeast two-hybrid analysis Yeast two-hybrid experiments were performed using the Matchmaker Gold Y2H system (Takara Bio) based on the manufacturer’s recommendations with modifications. Briefly, the S. cerevisiae strain Y2HGold was co-transformed with TRP1 -marked Gal4-BD and LEU2 -marked Gal4-AD plasmids, pGBKT7 and pGADT7, respectively. Co-transformed strains were cultured in SC-Leu-Trp medium at 30°C overnight, adjusted to an OD600 of 1, and serially diluted tenfold. Five microliters of each dilution was spotted onto SC-Leu-Trp, SC-Leu-Trp-His, or SC-Leu-Trp-His plates supplemented with 2 mM 3-Amino-1,2,4-triazole (3-AT, MilliporeSigma). Plates were incubated at 30°C and imaged daily for a minimum of four days. Funding National Science Foundation grant 2346844 (RD). National Institutes of Health grant R35GM160259 (RD). National Institutes of Health grant P20GM103636 (Project 2, RD). Oklahoma Center for Adult Stem Cell Research grant 231012 (RD). National Institutes of Health grant P30GM149376 (Bioinformatics and Pathway Core, KB). Author contributions Conceptualization: JR, MD, RD Methodology: JR, MD, MK, KB, RD Investigation: IP, JR, AM, MD, MK, RD Visualization: IP, JR, AM, RD Supervision: JR, MD, RD Writing—original draft: RD Writing—review & editing: JR, MD, RD Competing interests The authors declare no competing interests. Data and materials availability All data are available in the main text or the supplementary materials. SLAM-seq and ChEC-seq datasets have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE313013. Raw datasets from DIA and PRM mass spectrometry experiments have been deposited in the Zenodo repository ( https://doi.org/10.5281/zenodo.17966389 ). Reference genome assemblies and annotations for S. cerevisiae strain S288C (version R64-3-1) and S. pombe strain 972h- (version ASM294v2) were retrieved from the NCBI Datasets repository (GCF 000146045.2 and GCF 000002945.1, respectively). BED files for S. cerevisiae and S. pombe used in SLAM-DUNK were retrieved from the Zenodo repository ( https://doi.org/10.5281/zenodo.10714018 ). Acknowledgments We thank Linda Thompson, Dean Dawson, Christopher Sansam and Karen Arndt for helpful comments, Steven Hahn for plasmids, and Stephen Buratowski for anti-Bdf1 antibody. We thank the OMRF Research Computing Services, Clinical Genomics Center, and Center for Biomedical Data Sciences for contributions to this work. Funder Information Declared National Science Foundation , 2346844 Oklahoma Center for Adult Stem Cell Research , 231012 National Institutes of Health , P20GM103636 , R35GM160259 , P30GM149376 Footnotes Additional experiments resulted in expanded Fig. 5, addition of Fig. 6 and several supplementary figures. The conclusions were changed accordingly. References 1. ↵ Filippakopoulos , P. et al. Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family . Cell 149 , 214 – 231 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Donczew , R. & Hahn , S . BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae . Elife 10 , e69619 ( 2021 ). OpenUrl CrossRef PubMed 3. ↵ Muhar , M. et al. SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis . Science 360 , 800 – 805 ( 2018 ). OpenUrl Abstract / FREE Full Text 4. ↵ Winter , G. E. et al. BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment . Molecular Cell 67 , 5 – 18 .e19 ( 2017 ). OpenUrl CrossRef PubMed 5. ↵ Zheng , B. et al. Distinct layers of BRD4-PTEFb reveal bromodomain-independent function in transcriptional regulation . Molecular Cell 83 , 2896 – 2910 .e4 ( 2023 ). OpenUrl CrossRef PubMed 6. ↵ Fujisawa , T. & Filippakopoulos , P . Functions of bromodomain-containing proteins and their roles in homeostasis and cancer . Nature Reviews Molecular Cell Biology 18 , 246 – 262 ( 2017 ). OpenUrl CrossRef PubMed 7. ↵ Wang , N. , Wu , R. , Tang , D. & Kang , R . The BET family in immunity and disease . Signal Transduction and Targeted Therapy 6 , 1 – 22 ( 2021 ). OpenUrl CrossRef 8. ↵ Filippakopoulos , P. et al. Selective inhibition of BET bromodomains . Nature 468 , 1067 – 1073 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 9. Nicodeme , E. et al. Suppression of inflammation by a synthetic histone mimic . Nature 468 , 1119 – 1123 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 10. ↵ Shu , S. et al. Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer . Nature 529 , 413 – 417 ( 2016 ). OpenUrl CrossRef PubMed 11. ↵ Calder , J. , Nagelberg , A. , Luu , J. , Lu , D. & Lockwood , W. W . Resistance to BET inhibitors in lung adenocarcinoma is mediated by casein kinase phosphorylation of BRD4 . Oncogenesis 10 , 27 ( 2021 ). OpenUrl CrossRef PubMed 12. ↵ Sun , Y. et al. Epigenetic heterogeneity promotes acquired resistance to BET bromodomain inhibition in ovarian cancer . Am J Cancer Res 11 , 3021 – 3038 ( 2021 ). OpenUrl PubMed 13. ↵ Arnold , M. , Bressin , A. , Jasnovidova , O. , Meierhofer , D. & Mayer , A . A BRD4-mediated elongation control point primes transcribing RNA polymerase II for 3’-processing and termination . Mol Cell S1097-2765(21)00506–2 ( 2021 ) doi: 10.1016/j.molcel.2021.06.026 . OpenUrl CrossRef PubMed 14. ↵ Zheng , B. et al. BRD2 bridges TFIID and MOF-H4K16ac-containing nucleosomes to promote transcriptional initiation . Molecular Cell https://doi.org/10.1016/j.molcel.2025.12.010 ( 2025 ) doi: 10.1016/j.molcel.2025.12.010 . OpenUrl CrossRef 15. ↵ Matangkasombut , O. , Buratowski , R. M. , Swilling , N. W. & Buratowski , S . Bromodomain factor 1 corresponds to a missing piece of yeast TFIID . Genes Dev . 14 , 951 – 962 ( 2000 ). OpenUrl Abstract / FREE Full Text 16. ↵ Kanno , T. et al. BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones . Nat Struct Mol Biol 21 , 1047 – 1057 ( 2014 ). OpenUrl CrossRef PubMed 17. ↵ Lambert , J.-P. et al. Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains . Mol Cell 73 , 621 – 638 .e17 ( 2019 ). OpenUrl CrossRef PubMed 18. ↵ Matangkasombut , O. & Buratowski , S . Different sensitivities of bromodomain factors 1 and 2 to histone H4 acetylation . Mol. Cell 11 , 353 – 363 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 19. ↵ Slaughter , M. J. et al. HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies . Cell Rep 34 , 108638 ( 2021 ). OpenUrl CrossRef PubMed 20. ↵ Kayne , P. S. et al. Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast . Cell 55 , 27 – 39 ( 1988 ). OpenUrl CrossRef PubMed Web of Science 21. ↵ Ranish , J. A. et al. The study of macromolecular complexes by quantitative proteomics . Nat. Genet . 33 , 349 – 355 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 22. ↵ Zhang , S. & Roeder , R. G . Resistance of estrogen receptor function to BET bromodomain inhibition is mediated by transcriptional coactivator cooperativity . Nat Struct Mol Biol 1 – 15 ( 2024 ) doi: 10.1038/s41594-024-01384-6 . OpenUrl CrossRef 23. ↵ Rahman , S. et al. The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3 . Mol Cell Biol 31 , 2641 – 2652 ( 2011 ). OpenUrl Abstract / FREE Full Text 24. ↵ Zhang , Q. et al. Structural Mechanism of Transcriptional Regulator NSD3 Recognition by the ET Domain of BRD4 . Structure 24 , 1201 – 1208 ( 2016 ). OpenUrl CrossRef PubMed 25. ↵ Konuma , T. et al. Structural Mechanism of the Oxygenase JMJD6 Recognition by the Extraterminal (ET) Domain of BRD4 . Sci Rep 7 , 16272 ( 2017 ). OpenUrl CrossRef PubMed 26. ↵ Alerasool , N. , Leng , H. , Lin , Z.-Y. , Gingras , A.-C. & Taipale , M . Identification and functional characterization of transcriptional activators in human cells . Molecular Cell 82 , 677 – 695 .e7 ( 2022 ). OpenUrl CrossRef PubMed 27. Trojanowski , J. et al. Transcription activation is enhanced by multivalent interactions independent of phase separation . Molecular Cell 82 , 1878 – 1893.e10 ( 2022 ). OpenUrl CrossRef PubMed 28. ↵ Xing , E. et al. Development of Murine Leukemia Virus Integrase-Derived Peptides That Bind Brd4 Extra-Terminal Domain as Candidates for Suppression of Acute Myeloid Leukemia . ACS Pharmacol. Transl. Sci . 4 , 1628 – 1638 ( 2021 ). OpenUrl PubMed 29. ↵ Huang , Q. et al. Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo . J. Med. Chem . 67 , 6658 – 6672 ( 2024 ). OpenUrl PubMed 30. ↵ Wu , S.-Y. , Lee , A.-Y. , Lai , H.-T. , Zhang , H. & Chiang , C.-M . Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting . Mol Cell 49 , 843 – 857 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 31. ↵ Malvezzi , F. et al. Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins . Commun Biol 4 , 1 – 13 ( 2021 ). OpenUrl PubMed 32. ↵ Sawa , C. et al. Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2 . Mol. Cell. Biol . 24 , 4734 – 4742 ( 2004 ). OpenUrl Abstract / FREE Full Text 33. ↵ Han , X. et al. Roles of the BRD4 short isoform in phase separation and active gene transcription . Nat Struct Mol Biol 27 , 333 – 341 ( 2020 ). OpenUrl CrossRef PubMed 34. ↵ Ridenour , J. B. & Donczew , R . An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae . BMC Methods 1 , 8 ( 2024 ). OpenUrl 35. ↵ Donczew , R. , Warfield , L. , Pacheco , D. , Erijman , A. & Hahn , S . Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA . eLife 9 , e50109 ( 2020 ). OpenUrl CrossRef PubMed 36. ↵ Zentner , G. E. , Kasinathan , S. , Xin , B. , Rohs , R. & Henikoff , S . ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo . Nature Communications 6 , 8733 ( 2015 ). OpenUrl PubMed 37. ↵ Jumper , J. et al. Highly accurate protein structure prediction with AlphaFold . Nature 596 , 583 – 589 ( 2021 ). OpenUrl CrossRef PubMed 38. ↵ Zhou , D. et al. FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling . Nucleic Acids Res 50 , 8700 – 8718 ( 2022 ). OpenUrl CrossRef PubMed 39. ↵ Peterson , A. C. , Russell , J. D. , Bailey , D. J. , Westphall , M. S. & Coon , J. J . Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics* . Molecular & Cellular Proteomics 11 , 1475 – 1488 ( 2012 ). OpenUrl PubMed 40. ↵ True , J. D. et al. The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization* . Journal of Biological Chemistry 291 , 15307 – 15319 ( 2016 ). OpenUrl Abstract / FREE Full Text 41. Viktorovskaya , O. et al. Essential histone chaperones collaborate to regulate transcription and chromatin integrity . Genes Dev . 35 , 698 – 712 ( 2021 ). OpenUrl Abstract / FREE Full Text 42. ↵ Connell , Z. , Parnell , T. J. , McCullough , L. L. , Hill , C. P. & Formosa , T . The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II . Nucleic Acids Res 50 , 784 – 802 ( 2022 ). OpenUrl CrossRef PubMed 43. ↵ Garcia-Gutierrez , P. , Mundi , M. & Garcia-Dominguez , M . Association of bromodomain BET proteins with chromatin requires dimerization through the conserved motif B . Journal of Cell Science 125 , 3671 – 3680 ( 2012 ). OpenUrl Abstract / FREE Full Text 44. ↵ Stortz , M. , Presman , D. M. & Levi , V . Transcriptional condensates: a blessing or a curse for gene regulation? Commun Biol 7 , 1 – 10 ( 2024 ). OpenUrl PubMed 45. ↵ Sabari , B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control . Science 361 , ( 2018 ). 46. ↵ Gilan , O. et al. Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation . Science 368 , 387 – 394 ( 2020 ). OpenUrl Abstract / FREE Full Text 47. ↵ Christiano , R. , Nagaraj , N. , Fröhlich , F. & Walther , T. C . Global Proteome Turnover Analyses of the Yeasts S. cerevisiae and S. pombe . Cell Reports 9 , 1959 – 1965 ( 2014 ). OpenUrl PubMed 48. Mahendrawada , L. , Warfield , L. , Donczew , R. & Hahn , S . Low overlap of transcription factor DNA binding and regulatory targets . Nature 642 , 796 – 804 ( 2025 ). OpenUrl PubMed 49. ↵ Roy , K. & Chanfreau , G . Stress-Induced Nuclear RNA Degradation Pathways Regulate Yeast Bromodomain Factor 2 to Promote Cell Survival . PLOS Genetics 10 , e1004661 ( 2014 ). OpenUrl 50. ↵ Brachmann , C. B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications . Yeast 14 , 115 – 132 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 51. Warfield , L. et al. Transcription of Nearly All Yeast RNA Polymerase II-Transcribed Genes Is Dependent on Transcription Factor TFIID . Mol Cell 68 , 118 – 129 .e5 ( 2017 ). OpenUrl CrossRef PubMed 52. ↵ Sikorski , R. S. & Hieter , P . A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae . Genetics 122 , 19 – 27 ( 1989 ). OpenUrl Abstract / FREE Full Text 53. ↵ Chen , S. , Zhou , Y. , Chen , Y. & Gu , J. fastp: an ultra-fast all-in-one FASTQ preprocessor . Bioinformatics 34 , i884 – i890 ( 2018 ). OpenUrl CrossRef PubMed 54. ↵ Neumann , T. et al. Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets . BMC Bioinformatics 20 , 258 ( 2019 ). OpenUrl CrossRef PubMed 55. ↵ Love , M. I. , Huber , W. & Anders , S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biology 15 , 550 ( 2014 ). OpenUrl CrossRef PubMed 56. ↵ Mahendrawada , L. , Warfield , L. , Donczew , R. & Hahn , S . Low overlap of transcription factor DNA binding and regulatory targets . Nature 1 – 9 ( 2025 ) doi: 10.1038/s41586-025-08916-0 . OpenUrl CrossRef PubMed 57. ↵ Warfield , L. , Donczew , R. , Mahendrawada , L. & Hahn , S . Yeast Mediator facilitates transcription initiation at most promoters via a Tail-independent mechanism . Molecular Cell 82 , 4033 – 4048.e7 ( 2022 ). OpenUrl CrossRef PubMed 58. ↵ Möller , M. , Ridenour , J. B. , Wright , D. F. , Martin , F. A. & Freitag , M . H4K20me3 is important for Ash1-mediated H3K36me3 and transcriptional silencing in facultative heterochromatin in a fungal pathogen . PLOS Genetics 19 , e1010945 ( 2023 ). OpenUrl PubMed 59. ↵ Langmead , B. & Salzberg , S. L . Fast gapped-read alignment with Bowtie 2 . Nature Methods 9 , 357 – 359 ( 2012 ). OpenUrl PubMed 60. ↵ Danecek , P. et al. Twelve years of SAMtools and BCFtools . Gigascience 10 , giab008 ( 2021 ). OpenUrl CrossRef PubMed 61. ↵ Ramírez , F. et al. deepTools2: a next generation web server for deep-sequencing data analysis . Nucleic Acids Research 44 , W160 – W165 ( 2016 ). OpenUrl CrossRef PubMed 62. ↵ Quinlan , A. R. & Hall , I. M . BEDTools: a flexible suite of utilities for comparing genomic features . Bioinformatics 26 , 841 – 842 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 63. ↵ Kent , W. J. , Zweig , A. S. , Barber , G. , Hinrichs , A. S. & Karolchik , D . BigWig and BigBed: enabling browsing of large distributed datasets . Bioinformatics 26 , 2204 – 2207 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 64. ↵ Heinz , S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities . Mol Cell 38 , 576 – 589 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 65. ↵ Park , D. , Morris , A. R. , Battenhouse , A. & Iyer , V. R . Simultaneous mapping of transcript ends at single-nucleotide resolution and identification of widespread promoter-associated non-coding RNA governed by TATA elements . Nucleic Acids Res 42 , 3736 – 3749 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 66. ↵ Krystkowiak , I. & Davey , N. E . SLiMSearch: a framework for proteome-wide discovery and annotation of functional modules in intrinsically disordered regions . Nucleic Acids Research 45 , W464 – W469 ( 2017 ). OpenUrl CrossRef PubMed 67. ↵ The Alliance of Genome Resources Consortium . Updates to the Alliance of Genome Resources central infrastructure . Genetics 227 , iyae049 ( 2024 ). OpenUrl CrossRef PubMed 68. ↵ Mészáros , B. , Erdős , G. & Dosztányi , Z . IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding . Nucleic Acids Res 46 , W329 – W337 ( 2018 ). OpenUrl CrossRef PubMed 69. ↵ Piovesan , D. , Monzon , A. M. & Tosatto , S. C. E . Intrinsic protein disorder and conditional folding in AlphaFoldDB . Protein Science 31 , e4466 ( 2022 ). OpenUrl CrossRef PubMed 70. ↵ Evans , R. et al. Protein complex prediction with AlphaFold-Multimer . 2021.10.04.463034 Preprint at doi: 10.1101/2021.10.04.463034 ( 2022 ). OpenUrl Abstract / FREE Full Text 71. ↵ Berman , H. M. et al. The Protein Data Bank . Nucleic Acids Res 28 , 235 – 242 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 72. ↵ Meng , E. C. et al. UCSF ChimeraX: Tools for structure building and analysis . Protein Science 32 , e4792 ( 2023 ). OpenUrl CrossRef PubMed 73. ↵ Mirdita , M. et al. ColabFold: making protein folding accessible to all . Nat Methods 19 , 679 – 682 ( 2022 ). OpenUrl CrossRef PubMed 74. ↵ Dunbrack , R. L. Rēs ipSAE loquunt: What’s wrong with AlphaFold’s ipTM score and how to fix it . 2025.02.10.637595 Preprint at doi: 10.1101/2025.02.10.637595 ( 2025 ). OpenUrl Abstract / FREE Full Text 75. ↵ Hekkelman , M. L. , Salmoral , D. Á. , Perrakis , A. & Joosten , R. P . DSSP 4: FAIR annotation of protein secondary structure . Protein Science 34 , e70208 ( 2025 ). OpenUrl PubMed 76. ↵ Cuevas-Bermúdez , A. , Garrido-Godino , A. I. & Navarro , F . A novel yeast chromatin-enriched fractions purification approach, yChEFs, for the chromatin-associated protein analysis used for chromatin-associated and RNA-dependent chromatin-associated proteome studies from Saccharomyces cerevisiae . Gene Reports 16 , 100450 ( 2019 ). OpenUrl 77. ↵ de Jonge , W. J. , Brok , M. , Kemmeren , P. & Holstege , F. C. P . An Optimized Chromatin Immunoprecipitation Protocol for Quantification of Protein-DNA Interactions . STAR Protocols 1 , 100020 ( 2020 ). OpenUrl PubMed 78. ↵ Rindler , P. M. , Plafker , S. M. , Szweda , L. I. & Kinter , M . High Dietary Fat Selectively Increases Catalase Expression within Cardiac Mitochondria * . Journal of Biological Chemistry 288 , 1979 – 1990 ( 2013 ). OpenUrl Abstract / FREE Full Text 79. ↵ Demichev , V. , Messner , C. B. , Vernardis , S. I. , Lilley , K. S. & Ralser , M . DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput . Nat Methods 17 , 41 – 44 ( 2020 ). OpenUrl CrossRef PubMed 80. ↵ Pino , L. K. et al. The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics . Mass Spectrometry Reviews 39 , 229 – 244 ( 2020 ). OpenUrl CrossRef PubMed 81. Lin , Y.-J. et al. Solution structure of the extraterminal domain of the bromodomain-containing protein BRD4 . Protein Science 17 , 2174 – 2179 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 82. Wang , Y. et al. Coiled-coil networking shapes cell molecular machinery . MBoC 23 , 3911 – 3922 ( 2012 ). OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted January 16, 2026. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following The extra-terminal domain drives the role of BET proteins in transcription Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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