RB dependent transcriptional regulation at mitotic centromeres preserves genome stability

RB dependent transcriptional regulation at mitotic centromeres preserves genome stability | 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 RB dependent transcriptional regulation at mitotic centromeres preserves genome stability Elizabeth A Crowley , View ORCID Profile Amity L Manning doi: https://doi.org/10.1101/2025.06.04.657825 Elizabeth A Crowley 1 Department of Biology and Biotechnology, Worcester Polytechnic Institute , Worcester, MA, 01609 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amity L Manning 1 Department of Biology and Biotechnology, Worcester Polytechnic Institute , Worcester, MA, 01609 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amity L Manning For correspondence: almanning{at}wpi.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Transcripts derived from centromere repeats play a critical role in the localization and activity of kinetochore components during mitosis such that disruption of RNA polymerase II-dependent transcription compromises the fidelity of chromosome segregation. Here, we show that the retinoblastoma tumor suppressor protein (RB), a critical regulator of the G1/S cell cycle transition, additionally plays an important role in the regulation of centromere transcription during mitosis. We find that cells lacking RB experience increased RNA Polymerase II activity at mitotic centromeres and a corresponding increase in nascent RNA transcripts derived from centromere sequences. Together with high levels of centromere transcription and corresponding R-loop formation, RB-deficient cells exhibit centromere DNA breaks and local activation of ATR that correspond with increased centromere localization of Aurora B, destabilization of kinetochore-microtubule attachments, and an increase in anaphase defects. Importantly, reduction of DNA damage, ATR activity, and mitotic defects following inhibition of RNA Pol II, or targeted repression of centromere transcription through centromere tethering of Suv420H2, support that mitotic defects in RB-deficient cells are linked to centromere transcription. Introduction During cell division newly replicated chromosomes are segregated equally into two daughter cells, ensuring the faithful inheritance of genetic information. This process is dependent on a specialized region of the chromosome known as the centromere ( Brinkley & Stubblefield, 1966 ; Musacchio & Salmon, 2007 ) which is epigenetically defined by the presence of nucleosomes containing the histone variant Centromere Protein A (CENP-A) ( Earnshaw et al , 1986 ; Fachinetti et al , 2013 ; Palmer et al , 1991 ). Proteinaceous structures known as kinetochores are then assembled at each centromere where they govern the interaction between chromosomes and microtubules of the mitotic spindle. Long thought to be transcriptionally inert, we now appreciate that both the core centromere region and the pericentric flanking regions are actively transcribed by RNA polymerase II (RNAPII) ( Chan et al , 2012 ; Ideue et al , 2014 ; Rošić & Erhardt, 2016 ; Wong et al , 2007 ). Transcriptional activity at centromeres is a conserved property among species ( Melters et al , 2013 ; Talbert et al , 2018 ) and has an important role in regulating centromere and kinetochore composition (reviewed in ( Perea-Resa & Blower, 2018 )). A key function of centromere transcription is to promote chromatin-remodeling that is permissive for loading of CENP-A-containing nucleosomes during the G 1 phase of the cell cycle ( Bobkov et al , 2018 ; Dunleavy et al , 2011 ; Jansen et al , 2007 ). However, the transcription of centromeres is not limited to interphase cells and RNAPII is active at mitotic centromeres ( Chan et al ., 2012 ). During cell division, centromere transcripts act as molecular tethers that can recruit and activate key centromere and kinetochore proteins, including CENP-C ( Bury et al , 2020 ; McNulty et al , 2017 ; Politi et al , 2002 ) which functions to stabilize CENP-A retention ( Falk et al , 2015 ; Guo et al , 2017 ; McNulty et al ., 2017 ; Watanabe et al , 2019 ), AurB kinase, a member of the chromosomal passenger complex (CPC) that modulates kinetochore-microtubule attachments ( Blower, 2016 ; Ferri et al , 2009 ; Jambhekar et al , 2014 ; Quénet & Dalal, 2014 ), and Shugoshin 1, which regulates centromere cohesion ( Chen et al , 2021 ; Liu et al , 2015 ). The formation of transcription-dependent R loops, where ssDNA is displaced as the transcription machinery moves along the template strand, also recruits the ataxia-telangiectasia and Rad3-related protein kinase (ATR) ( Matos et al , 2020 ). During mitosis, ATR functions, in part, to active Aurora B kinase (AurB) locally at the centromere ( Kabeche et al , 2018 ). Together, ATR and AurB dynamically regulate kinetochore microtubules to promote accurate chromosome segregation during cell division. The variety of ways in which transcriptional activity at the centromere contributes to mitotic fidelity indicates that genetic or epigenetic changes that alter the accessibility of the centromere to transcription factors or otherwise perturb the level of centromere transcription during mitosis, have the potential to impact both kinetochore composition and the fidelity of chromosome segregation. Consistent with this model, two functionally relevant marks of transcriptionally silent heterochromatin, trimethylation of lysine 9 on Histone H3 (H3K9me3) and trimethylation of lysine 20 on Histone H4 (H4K20me3), are highly enriched at pericentromeric regions and yet are restricted from centromeres ( Agredo & Kasinski, 2023 ; Schotta et al , 2004 ). Experimental manipulations that permit the spread of heterochromatin into the centromere corrupt deposition of the centromere specific CENPA- containing nucleosomes and lead to chromosome segregation errors ( Martins et al , 2020 ; Sidhwani & Straight, 2023 ). Interestingly, the absence or loss of heterochromatin from the pericentromere can also compromise the accuracy of chromosome segregation ( Herlihy et al , 2021 ; Martins et al , 2016 ; Martins et al ., 2020 ). The retinoblastoma tumor suppressor protein, RB, physically interacts with the enzymes that place the heterochromatic marks H3K9me3 (Suv39) and H4K20me3 (Suv420h2) ( Sanidas et al , 2019 ). RB-dependent recruitment of Suv420h2 is relevant for establishment of pericentric H4K20me3 ( Gonzalo & Blasco, 2005 ; Gonzalo et al , 2005 ) and loss of RB results in decreased H4K20me3 and cohesin complex (a reader of H4K20me3) at pericentromeres (Gonzalo et al , 2005( Manning et al , 2010 ; Manning et al , 2014 ). Here we demonstrate that high levels of centromere transcription and corresponding mis-regulation of mitotic kinases underlie centromere damage and mitotic errors that result from loss of RB. Furthermore, we show that suppression of mitotic transcription, centromere-targeted restoration of epigenetic silencing, or titration of kinase activity are sufficient to restore mitotic fidelity in cells lacking the RB tumor suppressor. Together these findings indicate that epigenetic regulation of centromeres is a dynamic and targetable process by which to modulate the accuracy of chromosome segregation. Results RB loss promotes RNA Polymerase II-dependent centromere transcription To determine whether loss of RB impacts transcription during mitosis, we first examined cells with and without RB depletion for evidence of RNA polymerase II (RNAPII) activity. Using human telomerase reverse transcriptase (hTERT)- immortalized retinal pigment epithelial cells (RPE-1) engineered to carry an inducible RB-targeting shRNA construct (hTERT-RPE-1 shRB), we treated cells with 2 µg/mL of doxycycline for 48 hours to induce RB depletion ( Figure 1A ). Nocodazole arrested mitotic cells were collected and chromosome spreads were prepared for immunofluorescence analysis of total RNAPII and active RNAPII (phosphorylated at Serine 2, which is indicative of elongating polymerase) ( Figure 1B ). RNAPII activity has previously been described at mitotic centromeres ( Chan et al, 2012 ). Consistent with these reports, hTERT-RPE-1 cells were positive for both total (RNAPII) and active (RNAPII pS2) RNA polymerase II. Using quantitative measures of RNAPII and RNAPII pS2 signal intensity across anti-centromere-antigen (ACA)-labelled kinetochores, we observe that while depletion of RB does not affect the total pool of RNAPII at mitotic centromeres, it does lead to a greater than 4-fold increase in RNAPII pS2, indicating that cells lacking RB experience an increase in transcriptional activity at mitotic centromeres ( Figure 1C ). Download figure Open in new tab Figure 1: RB depletion increases RNAPII activity and transcription at the centromere. A) RB depletion via shRNA (shRB) in hTERT-RPE-1 cells was verified by Western blot. B) Representative images of mitotic spreads stained for total RNAPII (pink), active RNAPII (pS2, green), ACA (red), and DNA (DAPI, blue). Scale bar = 2μM. C) Quantification of RNAPII and RNAPIIpS2 signal intensity across ACA-labelled centromeres. A minimum of 90 kinetochore pairs were measured (3/cell for 30 cells), for each of 3 biological replicates. D) Schematic representation of protocol to label nascent mitotic RNA. E) qPCR analysis of nascent (EU-labelled RNAs) and total RNA in control and RB-depleted cells. Individual replicates are indicated by different colors, statistical analyses were performed between averages of biological replicates; *: p< 0.05, **: p<0.01; ***: p<0.001, ****: p<0.001. To verify that RNA polymerase activity during mitosis corresponds with an increase in the synthesis of centromere transcripts, we first pulsed nocodazole-arrested mitotic cells with 5-ethynyl uridine (5-EU) for one hour. 5-EU is an analogue of uridine that is incorporated during RNA synthesis. Using click chemistry to link EU-labeled RNAs to biotin, followed by streptavidin bead pull down, newly synthesized mitotic RNAs were isolated from bulk cellular RNA ( Figure 1D ). Quantitative real-time PCR was used to perform comparative analysis of bulk and nascent centromere transcript levels from control and RB depleted mitotic hTERT-RPE-1 cells ( Figure 1E ). We find that mitotic cells lacking RB exhibit a significant increase in newly synthesized (nascent) centromere and pericentromere transcripts. A similar increase in transcript level is seen regardless of whether the sequence of the transcript analyzed is unique to a single chromosome (i.e. D7Z1/2 and D17Z1/b: centromere/pericentromere of chromosome 7 and 17, respectively) or common to multiple chromosomes (i.e. P82H: centromere and K111: pericentromere), suggesting an underlying, wide-spread dysregulation of transcriptional control at mitotic centromeres and pericentromeres in cells lacking RB. RB loss promotes DNA damage and ATR activation at mitotic centromeres Unzipping of the DNA double stranded helix while the RNA polymerase reads and transcribes the nascent RNA leads to the formation of a transient 3-stranded DNA-RNA hybrid structure known as an R loop ( Hamperl & Cimprich, 2016 ; Santos-Pereira & Aguilera, 2015 ; Thomas et al , 1976 ). Although a normal consequence of RNA transcription, these hybrid structures are prone to both single stranded (ssDNA) and double stranded DNA (dsDNA) breaks ( Aguilera & García-Muse, 2012 ; Cohen et al , 2018 ). Perturbations in the balance between formation and resolution of R-loops contribute to DNA damage and genomic instability ( Costantino & Koshland, 2018 ). Given our observation that loss of RB increases mitotic centromere transcription ( Figure 1 ), and increased transcription is known to enhance R-loop- induced DNA damage ( Crossley et al , 2019 ), we hypothesized that mitotic cells lacking RB may exhibit increased DNA damage at centromeres. To examine this possibility, hTERT-RPE-1 cells with and without induced expression of an RB-targeting hairpin (shRB) were arrested in mitosis for 4 hours, fixed and stained for the canonical DNA damage marker γH2AX ( Figure 2A ), and number of damage foci per cell quantified. This analysis revealed that cells lacking RB exhibit an increase in the fraction of mitotic cells exhibiting DNA damage (classified as 5 or more γH2AX foci in an individual cell; Figure 2B ). Complementary approaches to examine γH2AX foci on mitotic chromosome spreads from control and RB-depleted cells indicate that many of these damage foci are localized proximal to centromeres ( Figure 2C & 2D ). Importantly, the increase in mitotic DNA damage following RB depletion is dependent on ongoing transcription during mitosis, as treatment of mitotic cells with the RNAPII inhibitor α-amanitin (administered concurrent with nocodazole-induced arrest) restored the proportion of cells exhibiting DNA damage to that seen in control cells ( Figure 2A and 2B ). Download figure Open in new tab Figure 2: RB loss leads to centromere proximal DNA breaks. A & B) Representative images and quantification of γH2AX foci in mitotic hTERT-RPE-1 cells with or without induced shRNA-targeted depletion of RB (shRB) cells and/or treatment with α-amanitin (50μg/ml) during mitosis. Scale bar = 5μm, error bars represent standard deviation between biological replicates. C & D) Representative images and quantification of centromere-proximal γH2AX signal (green) in metaphase spreads co-stained for ACA (red) and DNA (DAPI, blue). A minimum of 90 kinetochore pairs were measured (3/cell for 30 cells), for each of 3 biological replicates. E) Western blot validation of RB loss in CRISPR-RB knockout hTERT-RPE cells (RB KO ). F & G) Representative images and quantification of cenFISH probe labelling in WT and RB KO cells following treatment (or not) with Exonuclease III. A minimum of 180 kinetochore pairs were measured (6/cell for 30 cells), for each of 3 biological replicates. In panels C& F white arrowheads or boxes indicate chromosomes represented in enlargements, scale bars are 2μm. Individual replicates are indicated by different colors, statistical analyses were performed between averages of biological replicates; **: p<0.01, ***: p<0.001. While transcription-induced R-loops can lead to both single strand and double strand DNA breaks, γH2AX most efficiently labels double strand breaks ( Rogakou et al , 1998 ; Sedelnikova et al , 2002 ). Therefore, to more comprehensively assess the extent to which RB-deficient cells acquire centromere and pericentromere nicks or breaks, we employed a fluorescent in situ hybridization (FISH) approach, termed exoFISH. In this assay, non-denaturing conditions limit centromere-targeting FISH probes to hybridize only when single-stranded DNA sequences are revealed following in vitro digestion with exonuclease III (ExoIII). Since ExoIII gains access via nicks in the DNA backbone, preexisting ss or dsDNA breaks enable ExoIII-dependent digestion ( Rogers & Weiss, 1980 ; Saayman et al , 2023b ) and a corresponding increase in FISH probe hybridization. Using a probe that specifically targets the centromere-localized CENP-B binding sites present on most chromosomes, we examined mitotic chromosome spreads from both control (WT) and RB knockout hTERT-RPE cells (RB KO ) ( Nicolay et al , 2015 ) for evidence of centromere-proximal breaks ( Figure 2E ). The specificity and sensitivity of this assay in revealing the presence of pre-existing DNA breaks are supported by negative controls in which both WT and RB KO show low levels of centromere FISH probe hybridization in the absence of ExoIII treatment and positive controls in which WT and RB KO cells pretreated with the DNA nicking enzyme Nt.BsmAI exhibit comparable levels of centromere FISH probe hybridization following ExoIII treatment ( Figure 2F & 2G , Supplemental Figure 1A & 1B ). Using this assay, we find that centromeres in RB KO cells are more susceptible to exonuclease activity than control cells ( Figure 2G ). This increase in centromere FISH probe accessibility is indicative of increased damage at or near mitotic centromeres. Download figure Open in new tab Supplemental Figure 1: DNA breaks at the centromere can be detected through exoFISH. A & B) Representative images and quantification of centromeres in metaphase spreads of control (WT) and RB knockout hTERT-RPE-1 cells (RB KO ) cells. Cells are labeled with a cenFISH probe following treatment, or not, with Nt.BsmAI and Exonuclease III. A minimum of 180 kinetochore pairs were measured (6/cell for 30 cells), for each of 3 biological replicates. Individual replicates are indicated by different colors, statistical analyses were performed between averages of biological replicates; *** p<0.001, ****: p<0.0001. R-loops recruit and activate the DNA damage response element ATR ( Kabeche et al ., 2018 ). During mitosis, ATR activity at centromeres stimulates AurB kinase to promote kinetochore-microtubule turnover ( Cimini et al , 2006 ). In agreement with published reports ( Kabeche et al ., 2018 ), we find that ATR is recruited to mitotic centromeres in both control and RB-depleted cells ( Figure 3A & 3B ). However, consistent with observations that mitotic cells lacking RB exhibit high levels of transcription-dependent DNA damage ( Figure 2 ), ATR activity at the mitotic centromere (as measured by the presence of the autophosphorylation mark pATR-T1989) is increased following RB depletion ( Figure 3A and Figure 3B ). We additionally observe a comparable increase in recruitment of AurB kinase to mitotic centromeres ( Supplemental Figure 2 ). Importantly, the increase in ATR activity and AurB localization are transcription-dependent, as treatment with α-amanitin reduces centromere-localized pATR and AurB to levels seen in control cells ( Figure 3A and 3B , Supplemental Figure 2 ). AurB kinase promotes kinetochore-microtubule turnover, such that increased AurB localization at the mitotic centromere enhances microtubule release and results in mitotic errors ( Cimini et al ., 2006 ; Knowlton et al , 2006 ; Muñoz-Barrera & Monje-Casas, 2014 ) Consistent with this, we and others find that RB-deficient cells exhibit a high rate of mitotic segregation errors ( Figure 3C & 3D ; ( Coschi et al , 2010 ; Hernando et al , 2004 ; Manning et al ., 2010 ). In agreement with a model whereby increased transcription/high levels of R loops activate ATR and in turn enhance AurB activity, anaphase defects in RB-deficient cells are similarly suppressed by either RNAPII (α-amanitin) or ATR (VE-821) inhibition ( Figure 3C & 3D ). Download figure Open in new tab Supplemental Figure 2: Aurora B localization is increased following RB loss. A & B) Representative images and quantification of centromere-localized Aurora B (green) in hTERT-RPE-1 cells co-stained for ACA (red), and DNA (DAPI, blue) following shRNA targeted depletion of RB (shRB) and/or treatment with α-amanitin (50μg/ml). Insets are of individual kinetochore pairs at 3X magnification. A minimum of 90 kinetochore pairs were measured (3/cell for 30 cells), for each of 3 biological replicates. Scale bar is 5μm. Individual replicates are indicated by different colors, statistical analyses were performed between averages of biological replicates; ***: p<0.001. Download figure Open in new tab Figure 3: Transcription-dependent ATR activation compromises mitotic fidelity following loss of RB. A & B) Representative images and quantification of total (pink) and phosphorylated (T1989; green) ATR at mitotic ACA-labeled (red) centromeres in control and RB-depleted hTERT-RPE-1 cells. Cells were untreated or treated with the RNA polymerase II inhibitor α-amanitin (50μg/ml). A minimum of 90 kinetochore pairs were measured (3/cell for 30 cells), for each of 3 biological replicates. C & D) Representative images and quantification of anaphase defects in hTERT-RPE-1 cells treated with either a non-targeting control (S) or RB-specific (R) siRNA and subsequently treated with ATR inhibitor (VE-821, 10μM) for 1hr, α-amanitin (50μg/ml) for 4hrs. A minimum of 50 cells were scored per condition for each of 3 biological replicates. White arrowheads indicate lagging chromosomes, Scale bars are 2μm. Error bars represent standard deviation between biological replicates and statistical analyses were performed between averages of biological replicates, ****: p<0.0001. Epigenetic silencing of centromeres suppresses changes that result from RB loss Pericentromeres are enriched with heterochromatin-promoting histone modifications including H3K9me3 and H4K20me3 ( Jeppesen et al , 1992 ; Peters et al , 2001 ; Rea et al , 2000 ; Rice et al , 2003 ; Schotta et al ., 2004 ). The RB protein has been shown to physically and functionally interact with a number of chromatin modifiers, including the H4K20 methyltransferase Suv420H2 ( Gonzalo et al ., 2005 ; Isaac et al , 2006 ; Siddiqui et al , 2007 ), such that loss of RB leads to decreased Suv420H2 and H4K20me3 enrichment at pericentromeres and telomeres ( Gonzalo et al ., 2005 ; Isaac et al ., 2006 ). Given the transcriptionally repressive role of H4K20me3 ( Gonzalo et al ., 2005 ; Kourmouli et al , 2005 ; Schotta et al ., 2004 ; Sullivan & Karpen, 2004 ) and our observation that RB loss leads to transcriptional upregulation of mitotic centromeres, we sought to explore whether loss of Suv420H2 enrichment may underlie centromere deregulation when RB is lost or depleted. To this end, we employed a previously established centromere-tethering system to anchor Suv420H2-GFP, via fusion to the DNA binding domain of centromere protein CENP-B, to centromeres ( Herlihy et al ., 2021 ). Using doxycycline-induced expression of a cen-Suv420H2-GFP fusion protein ( Figure 4A ), we first examined the impact of centromere-tethered Suv420-GFP (cenSuv) on mitotic centromere transcription in control and RB-depleted cells. As described above, nascent centromere RNAs were labelled with 5-EU, isolated, and quantified using qPCR. These data revealed that mitotic centromere transcription, which is increased following RB depletion alone, is reduced by concurrent tethering of Suv420H2 to the centromere ( Figure 4B ). Download figure Open in new tab Figure 4: Decreasing mitotic centromere transcription reduces centromere breaks. A) Western blot analysis confirmation of RB knockdown and Suv420H2 overexpression in hTERT-RPE + inducible cen-Suv420H2-GFP cells treated with either a non-targeting control (S) or RB specific (R) siRNA, with or without induction of cen-Suv420H2-GFP expression. B) qPCR analysis of nascent (5-EU-labeled RNA) and total RNA transcribed from a representative centromere (D7Z1) and pericentromere (D7D2) in mitotic cells. C) Representative images and quantification of cenFISH signal in control and RB-depleted cells with or without Exonuclease III treatment. A minimum of 180 kinetochore pairs were measured (6/cell for 30 cells), for each of 3 biological replicates. Scale bars are 5μm for whole spread panels and 2μm for individual chromosome enlargement. Individual replicates are indicated by different colors, statistical analyses were performed between averages of biological replicates; *: p< 0.05, **: p<0.01; ***: p<0.001. Treatment with α-amanitin indicates that increased centromere damage, ATR activation, and increased centromere AurB localization that occur following RB loss are dependent on RNA polymerase II activity ( Figure 2 , Figure 3 ). However, α-amanitin treatment alone cannot discern between a role for centromere transcription and more general transcriptional deregulation that may occur throughout the genome when RB activity is lost. Therefore, to more explicitly test the role of centromere regulation on these attributes in RB-deficient cells, we performed exoFISH, as described above, on control and siRB-depleted cells with and without expression of centromere-tethered Suv420H2-GFP. Upon induction of cen-Suv420H2-GFP the high level of ExoIII-dependent cenFISH signal seen in siRB depleted cells was reduced ( Figure 4C & 4D ; Supplemental Figure 3 ). Download figure Open in new tab Supplemental Figure 3: Validation of centromeric DNA break detection using exoFISH in cen-Suv420H2-GFP cells. A) qPCR analysis to validate RB depletion in hTERT-RPE + cen-Suv420-GFP cells treated with either a non-targeting control (S) or RB specific (R) siRNA, with or without induction of centromere tethered Suv420H2 expression. B & C) Representative images and quantification of centromeres in metaphase spreads. Cells are labeled with a cenFISH probe following treatment, or not, with Nt.BsmAI and Exonuclease III. A minimum of 180 kinetochore pairs were measured (6/cell for 30 cells), for each of 3 biological replicates. Scale bar is 5μm for whole spread images and 2μm for individual chromosomes. Individual replicates are indicated by different colors, statistical analyses were performed between averages of biological replicates; ** p<0.01; ****: p<0.0001. We next employed quantitative immunofluorescence to assess centromere levels of AurB kinase and anaphase defects following induced centromere tethering of Suv420h2-GFP in control and RB-depleted cells. Similar to RB depletion strategies described above ( Supplemental Figure 2 ), siRB-treated cells exhibit an increase in the staining intensity of AurB at centromeres ( Figure 5A & 5B ) and an increase in anaphase defects compared to control cells treated with an si-Scramble sequence ( Figure 5C & 5D ). Consistent with suppression of both centromere transcription and centromere-proximal DNA damage described above, centromere tethering of Suv420H2-GFP in siRB cells reduced both centromere-localization of AurB ( Figure 5A and 5B ) and anaphase defects to levels comparable to that observed in cells treated with a scrambled siRNA control alone ( Figure 5C & 5D ). Download figure Open in new tab Figure 5: Suppression of transcription at the centromere limits Aurora B localization and reduces lagging chromosomes. A & B) Representative images and quantification of centromere-localized Aurora B (green) in hTERT-RPE + cen-Suv420H2-GFP cells treated with either a non-targeting control (S) or RB specific (R) siRNA, with or without induction of centromere tethered Suv420H2 and co-stained for ACA (red), GRF (white) and DNA (DAPI, blue). Insets are of individual kinetochore pairs at 3X magnification. A minimum of 90 kinetochore pairs were measured (3/cell for 30 cells), for each of 3 biological replicates. C) Representative images and quantification of anaphase defects in cells stained for ACA (red), tubulin (green), and DNA (DAPI, blue). A minimum of 50 cells were scored per condition for each of 3 biological replicates. Scale bars are 5μm. Error bars represent standard deviation between biological replicates. Individual replicates are indicated by different colors, statistical analyses were performed between averages of biological replicates; *: p< 0.05, **: p<0.01; ***: p<0.001, ****: p<0.0001. E) Model proposing how RB-dependent regulation of centromere transcription promotes mitotic fidelity. Dark blue text represents experimental readouts described in this study, red font represents experimental manipulations used to test/establish relationships. Discussion Here we show that loss of the RB tumor suppressor permits increased RNA polymerase II activity at mitotic centromeres ( Figure 1 ). The high temporal resolution afforded by first pulse labelling mitotic cells with 5-EU and then quantifying nascent RNA levels indicates that this feature of RB deficient cells corresponds with ongoing transcription at centromeres during mitosis ( Figures 1 and 4 ). Our complementary approaches to either visualize the DNA damage marker γH2AX or alternatively to exploit ss and dsDNA breaks with exonuclease treatment reveal an increase in centromere-proximal DNA breaks following RB loss ( Figures 2 and 4 ). We find that these breaks correspond with centromere-localized activation of ATR and AurB kinases ( Figures 3 , S2, 5). Consistent with previously described roles for ATR and AurB in the regulation of mitotic chromosome segregation, RB deficient cells exhibit an increase in anaphase defects ( Figures 3 and 5 ). Importantly, inhibition of bulk mitotic transcription (RNA polymerase II inhibition with α-amanitin) or centromere-specific transcription (through centromere-tethering of the heterochromatin-promoting enzyme Suv420H2) limit mitotic DNA damage ( Figures 2 and 4 ), reduce ATR and AurB kinase activity at centromeres ( Figures 3 and 5 ), and restore mitotic fidelity in RB-deficient cells ( Figures 3 and 5 ). These data support a model whereby high levels of centromere transcription renders cells lacking RB sensitive to DNA breaks and ATR activation, leading to increased AurB kinase localization and activity at centromeres. Together, these changes promote chromosome segregation errors and contribute to chromosome instability ( Figure 5E ). RB-dependent regulation of heterochromatic boundaries at centromeres is critical for mitotic fidelity Proteomics analysis has indicated that the RB tumor suppressor protein physically interacts with over 300 proteins, a significant portion of which are epigenetic modifying enzymes ( Sanidas et al ., 2019 ). Functional studies of the RB interactome suggest that RB may serve as a scaffold-moderating where in the genome and when in the cell cycle distinct epigenetic modifiers interact with chromatin (reviewed in ( Gonzalo & Blasco, 2005 )). Thus, by impacting the recruitment of enzymes that place transcriptionally repressive marks at centromeres (i.e. H3K27me3 by EZH2 ( Blais et al , 2007 ; Ishak et al , 2016 ), H3K9me3 by Suv39 ( Nielsen et al , 2001 ; Vandel et al , 2001 ), and H4K20me3 by Suv420h2 ( Gonzalo et al ., 2005 ; Manning et al ., 2014 )) RB is poised to limit centromere and pericentromere transcription. Consistent with work showing that disruption of RB interaction with EZH2 in mouse models deregulate transcriptional repression ( Ishak et al ., 2016 ) we demonstrate here that loss of RB permits high levels of transcriptional activity at the centromeres in mitotic human cells and leads to defects in chromosome segregation during cell division. Furthermore, rescue experiments that exploit mitotic specific (via short term α-amanitin treatment) or centromere-localized (via centromere-tethered Suv420h2/centromere-specific H4K20me3 enrichment) transcriptional repression to limit segregation defects indicate that mitotic errors following RB loss result from deregulation of centromere transcripts late in the cell cycle, and may not otherwise be dependent on global changes in protein expression that occur from loss of RB-dependent repression of E2F transcription factors earlier in the cell cycle. Balancing centromere transcription for genome and chromosome stability Prior studies have demonstrated that inhibition of RNAPII and the corresponding decrease in centromere transcription promote mitotic errors ( Chen et al ., 2021 ; McNulty et al ., 2017 ; Rošić et al , 2014 ). These studies implicate centromere transcription in the establishment of open chromatin that is conducive for CENP-A deposition in preparation for the subsequent cell cycle ( Bobkov et al ., 2018 ), and the transcripts themselves in recruiting and tethering critical kinetochore components ( Blower, 2016 ; Ferri et al ., 2009 ; Jambhekar et al ., 2014 ; Liu et al ., 2015 ; McNulty et al ., 2017 ; Quénet & Dalal, 2014 ). In the absence of centromere-derived transcripts the AurB kinase-containing chromosomal passenger complex (CPC) is not recruited to the kinetochore ( Blower, 2016 ; Jambhekar et al ., 2014 ). AurB kinase functions to destabilize kinetochore microtubules- a critical step in releasing improper attachments that form early in mitosis. In the absence of AurB activity, kinetochore-microtubule attachments are hyper stabilized and improper attachments persist, ultimately delaying biorientation and leading to segregation errors ( Abe et al , 2016 ; Broad et al , 2020 ; Hauf et al , 2003 ; Huang et al , 2018 ). In contrast to work showing that mitotic fidelity is sensitive to reduction of centromere transcription, de-repression and/or increased expression of repetitive sequences derived from or near centromeres, including LINE elements Satellite repeats, has been observed in various cancer contexts and is strongly correlated with poor patient prognosis ( McNulty et al ., 2017 ; Ting et al , 2011 ; Zhu et al , 2018 ; Zhu et al , 2011 ). While current studies have not yet discerned whether high levels of centromere transcription may be a driving force in tumorigenesis or merely a passenger that indicates widespread deregulation of transcriptional repression, recent molecular studies highlight several models whereby centromere transcription may directly impact genome stability and underlie cancer susceptibility (reviewed in ( Petermann et al , 2022 )). First, the process of transcription results in the generation of a DNA:RNA hybrid and displaced ssDNA structure known as an R loop. These structures are sensitive to both single strand and double strand DNA breaks and during S phase additionally pose an obstacle to replication that can promote replication stress and further DNA damage. To mitigate this risk, cells actively limit aberrant or excessive R-loop accumulation through the activity of DNA:RNA hybrid-specific endoribonuclease activity ( Lockhart et al , 2019 ). Second, the presence of R loops activates the DNA damage response. During mitosis, this includes the recruitment of the ATR kinase to centromeres. ATR activity leads to local activation of AurB kinase (via an ATR-Chk1 axis) ( Kabeche et al ., 2018 ). As described above, AurB kinase functions to destabilize kinetochore microtubule attachments ( Muñoz-Barrera & Monje-Casas, 2014 ) which can in turn compromise mitotic fidelity ( Crowley et al , 2022 ; González-Loyola et al , 2015 ; Muñoz-Barrera & Monje-Casas, 2014 ). Activated oncogenes have been described to increase transcription and R loop formation, leading to replication stress and genomic instability (reviewed in ( Petermann et al ., 2022 )). Our work additionally implicates the RB tumor suppressor, which is commonly lost or functionally inactivated across a broad range of cancer contexts ( Burkhart & Sage, 2008 ), as a regulator of mitotic centromere transcription. We propose that, through recruitment of key epigenetic modifying enzymes, RB functions to establish and/or maintain a heterochromatic boundary at centromeres that in turn limits centromere transcription and AurB activity to promote mitotic fidelity. A number of groups have described that loss of RB compromises mitotic fidelity ( Manning & Dyson, 2011 ). Data presented here are consistent with a model whereby, in the absence of RB, repressive marks near centromeres are reduced permitting excessive transcriptional activity. Aberrant R-loop formation and the corresponding recruitment of ATR kinase may then collaborate with transcript-dependent recruitment of AurB to destabilize kinetochore microtubules, concurrently leading to both centromere damage and whole chromosome segregation errors. Materials and Methods Cell culture hTERT immortalized RPE-1 RB KO (gift from the Dyson lab, Massachusetts General Hospital Cancer Center), RPE shRB ( Zamalloa et al , 2023 ), and RPE cen-suv420h2 GFP ( Herlihy et al ., 2021 ) cells were grown in Dulbecco’s Modified Essential Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin/streptomycin (Gibco). All cell lines were maintained at 37°C and 5% CO2. High resolution immunofluorescence imaging with DNA stain (DAPI, ThermoFisher) was used to monitor and confirm cell lines were free of Mycoplasma contamination. Over expression of cen-targeted suv420h2 GFP was achieved by treatment with 2µg/ml doxycycline for 16hrs. Inhibition of ataxia telangiectasia mutated and Rad3-related kinase (ATR) was achieved through treatment with VE-821 at a final concentration of 10µM (Sigma) for 1 hour. The inhibition of RNA polymerase II transcription was completed using a final concentration of 50ng/ml α-amanitin (Santa Cruz) for 4 hours. Depletion of RB1 was achieved through transient transfection with 50nM pool siRNAs (a pool of 4 siRNAs to RB1; siRNA-SMARTpool, Horzion Discovery) using RNAiMax transfection reagent (ThermoFisher) according to the manufacturer’s instructions. Transfection with a SMARTpool of four non-targeting siRNA sequences (Ambicon #AM4636) was used as a negative control for all depletions (Supplemental Table 1). Alternatively, cells were infected with a lentiviral construct containing a doxycycline inducible shRNA hairpin (tet-pLK0-Puro, Addgene #21915) for the targeted depletion of RB. Stable hairpin-expressing cells were selected with Puromycin for 10 days. Induced depletion was achieved by the addition of 2µg/ml doxycycline for a minimum of 48 hours. Immunoblotting Cell extracts were prepared using 2x Laemmli buffer (BioRad) with β-Mercaptoethanol (Sigma Aldrich). Protein concentrations were normalized to total cell number and samples run on an SDS-PAGE gel. Proteins were transferred to PVDF membrane (Millipore) and blocked in 1xTBST supplemented with 5% milk. Antibodies were diluted 1:1000 in 1xTBST/5% milk: DM1Aα (α-tubulin, Santa Cruz), RB1 (4H1, Cell Signaling), GFP (D5.1, Cell Signaling) and incubated at 4°C. Membranes were washed in 1xTBST, incubated for in corresponding HRP-conjugated secondary antibody (GE Healthcare), and developed using ProSignal Pico (Prometheus). Immunofluorescence and Metaphase Spreads Cultured cells were grown on coverslips, fixed, and stained for AurB and ACA as previously described in ( Kleyman et al , 2014 ). To visualize anaphase defects and monitor DNA damage in nocodazole arrested prometaphase cells were fixed with 4% paraformaldehyde for 20 minutes, extracted with 0.2% TritionX-100 in PBS for 10 minutes, and blocked with TBS + 1% BSA. Primary antibodies, ACA (1:500, Antibodies inc), DM1A (1:1000 Santa Crux), or γH2AX Ser139 (1:1000, Cell Signaling) were diluted in TBS +1% BSA. Secondary antibodies were diluted in 1% BSA + 0.2mg/ml DAPI and coverslips were mounted onto slides using Prolong Antifade Gold (Molecular Probes). Mitotic cells were collected via shake-off after treatment with 100ng/ml nocodazole (Selleckchem) for 4 hours and metaphase spreads prepared as in ( Keohane et al , 1996 ). Primary and secondary antibodies were diluted in KCM + 1% BSA. For staining of total (2B5,1:200 GeneTex) or phospho ATR (pT1989, 1:200 GeneTex) buffers were supplemented with a final concentration of 100nM Calyculin A (Millipore Sigma) ( Kabeche et al ., 2018 ). For total (F-12, Santa Cruz 1:200) or active RNAPII (pS2, Abcam 1:200) staining, buffers were supplemented with 100nM Calyculin A (Millipore Sigma), 40U/μL of RNasin (Promega) and kept on ice ( Chan et al ., 2012 ; Perea-Resa et al , 2020 ). Cells were then post fixed with 4% paraformaldehyde for 10 minutes prior to counter staining with TBS + 5% BSA + 0.2mg/ml DAPI for 30 minutes. Coverslips were mounted onto slides using Prolong Antifade Gold (Molecular Probes). Fixed cell images were captured using a Zyla sCMOS camera mounted on a Nikon Ti-E microscope, with a 60X Plan Apo oil immersion objective and 0.3 μm z-stacks. To assess centromeric protein levels, NIS-elements Advanced Research software was used to perform line scans in a single focal plane through individual ACA-stained kinetochore pairs where the area under the curve indicates the region of centromere/kinetochore-localized staining. γH2AX levels were assessed by counting the number of foci per cell. A cell was considered damaged if it had more than 5 foci per cell. For intensity measurements a minimum of 3 kinetochore pairs per 30 cells, per condition (90 kinetochore pairs/condition) were measured in each of 3 biological replicates. Anaphase defects were assessed in a minimum of 50 cells per condition for each of 3 biological replicates. exoFISH exoFISH was performed as described in ( Saayman et al , 2023a ) with the following modifications. Cells were first prepared and spread onto coverslips as in ( Ganem et al , 2009 ) Slides were then dried overnight at room temperature in the dark. The next day the slides were rehydrated in 1X PBS, treated with 0.5mg/ml RNaseA (NEB) for 10 minutes at 37°C in a humid chamber, washed with 1X PBS, then treated or not with 1 unit of Nt.BsmAI (NEB) for 2 hours at 37°C in a humid chamber. Slides were washed with 1X and treated or not with 200 mU/μL ExoIII diluted in 1X buffer supplied by the manufacturer for 1 hour then dehydrated overnight. To visualize breaks at the centromere slides were incubated in 0.5µM CENPB-binding site specific cenFISH probe (PNA Bio) for 3 hours at room temperature. Following incubation in hybridization wash buffers and subsequent dehydration, cover glass was then mounted onto slides using Prolong Antifade Gold. To quantify cenFISH signal, intensity values were measured within six 12x12 pixel boxes placed at centromeres and summed per cell for 30 cells per condition for each of 3 biological replicates. Fold change for each condition was calculated by normalizing to the RPE-1 without exoIII condition for that replicate. Experimental data were analyzed with a Student t-test or one-way ANOVA where appropriate. Individual measurements from experiments where multiple measurements were made per replicate are represented as SuperPlots, with individual replicates color-coded. Per-replicate averages and standard deviation between biological replicates are superimposed. All error bars represent standard deviation between biological replicates and statistically significant differences are labeled with *: p<0.05, **: p<0.01, ***: p<0.001, and **** p<0.0001. Quantification of Nascent and total RNA levels For quantification of nascent and total RNA levels mitotic cells were first isolated via mitotic shake off following incubation in 100 ng/ml nocodazole final concentration for 4 hours and subsequent treatment with 0.25µM 5-ethynyl uridine (5-EU, Vector Labs). Cells were processed according to the procedure outlined by the Click-iT™ Nascent RNA Capture Kit (Invitrogen). 2-5µg of RNA was removed prior to the remainder of the RNA undergoing the Click-iT™ reaction to be used for total RNA quantification and confirmation of RB knockdown. Total RNA was treated with DNaseI (NEB) before cDNA was synthesized from 1µg of total RNA using SuperScript IV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis for nascent RNA was performed according to the manufacturer’s instructions. Gene expression for centromeric and pericentromeric transcripts (Supplemental table 1) was determined using the ΔΔ cycle threshold method and normalized to GAPDH. Author Contributions ALM and EAC conceived and devised the study. EAC performed the experiments. ALM and EAC wrote the manuscript. Declaration of Interests Authors declare they have no conflict of interest. Acknowledgements The authors thank Nicole Hermance for technical support and critical evaluation of the manuscript. This work was supported by the American Cancer Society (RSG-21-066-01-CCG) to ALM. Footnotes This work was supported by the American Cancer Society (RSG-21-066-01-CCG). The authors declare no potential conflicts of interest. References ↵ Abe Y , Sako K , Takagaki K , Hirayama Y , Uchida KS , Herman JA , DeLuca JG , Hirota T ( 2016 ) HP1-Assisted Aurora B Kinase Activity Prevents Chromosome Segregation Errors . Dev Cell 36 : 487 – 497 OpenUrl CrossRef PubMed ↵ Agredo A , Kasinski AL ( 2023 ) Histone 4 lysine 20 tri-methylation: a key epigenetic regulator in chromatin structure and disease . Frontiers in Genetics Volume 14 – 2023 ↵ Aguilera A , García-Muse T ( 2012 ) R Loops: From Transcription Byproducts to Threats to Genome Stability . Molecular Cell 46 : 115 – 124 OpenUrl CrossRef PubMed Web of Science ↵ Blais A , van Oevelen CJ , Margueron R , Acosta-Alvear D , Dynlacht BD ( 2007 ) Retinoblastoma tumor suppressor protein–dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit . Journal of Cell Biology 179 : 1399 – 1412 OpenUrl Abstract / FREE Full Text ↵ Blower MD ( 2016 ) Centromeric Transcription Regulates Aurora-B Localization and Activation . Cell Rep 15 : 1624 – 1633 OpenUrl CrossRef PubMed ↵ Bobkov GOM , Gilbert N , Heun P ( 2018 ) Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation . J Cell Biol 217 : 1957 – 1972 OpenUrl Abstract / FREE Full Text Bosco N , Goldberg A , Zhao X , Mays JC , Cheng P , Johnson AF , Bianchi JJ , Toscani C , Di Tommaso E , Katsnelson L et al. ( 2023 ) KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres . Cell 186 : 1985 – 2001 .e1919 OpenUrl CrossRef PubMed ↵ Brinkley BR , Stubblefield E ( 1966 ) The fine structure of the kinetochore of a mammalian cell in vitro . Chromosoma 19 : 28 – 43 OpenUrl CrossRef PubMed Web of Science ↵ Broad AJ , DeLuca KF , DeLuca JG ( 2020 ) Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells . J Cell Biol 219 ↵ Burkhart DL , Sage J ( 2008 ) Cellular mechanisms of tumour suppression by the retinoblastoma gene . Nature Reviews Cancer 8 : 671 – 682 OpenUrl CrossRef PubMed Web of Science ↵ Bury L , Moodie B , Ly J , McKay LS , Miga KHH , Cheeseman IM ( 2020 ) Alpha-satellite RNA transcripts are repressed by centromere–nucleolus associations . eLife 9 : e59770 OpenUrl CrossRef PubMed ↵ Chan FL , Marshall OJ , Saffery R , Won Kim B , Earle E , Choo KHA , Wong LH ( 2012 ) Active transcription and essential role of RNA polymerase II at the centromere during mitosis . Proceedings of the National Academy of Sciences 109 : 1979 – 1984 OpenUrl Abstract / FREE Full Text ↵ Chen Y , Zhang Q , Teng Z , Liu H ( 2021 ) Centromeric transcription maintains centromeric cohesion in human cells . J Cell Biol 220 ↵ Cimini D , Wan X , Hirel CB , Salmon ED ( 2006 ) Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors . Curr Biol 16 : 1711 – 1718 OpenUrl CrossRef PubMed Web of Science ↵ Cohen S , Puget N , Lin YL , Clouaire T , Aguirrebengoa M , Rocher V , Pasero P , Canitrot Y , Legube G ( 2018 ) Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations . Nat Commun 9 : 533 OpenUrl CrossRef PubMed Contreras-Galindo R , Fischer S , Saha AK , Lundy JD , Cervantes PW , Mourad M , Wang C , Qian B , Dai M , Meng F et al. ( 2017 ) Rapid molecular assays to study human centromere genomics . Genome Res 27 : 2040 – 2049 OpenUrl Abstract / FREE Full Text ↵ Coschi CH , Martens AL , Ritchie K , Francis SM , Chakrabarti S , Berube NG , Dick FA ( 2010 ) Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive . Genes Dev 24 : 1351 – 1363 OpenUrl Abstract / FREE Full Text ↵ Costantino L , Koshland D ( 2018 ) Genome-wide Map of R-Loop-Induced Damage Reveals How a Subset of R-Loops Contributes to Genomic Instability . Mol Cell 71 : 487 – 497 .e483 OpenUrl CrossRef PubMed ↵ Crossley MP , Bocek M , Cimprich KA ( 2019 ) R-Loops as Cellular Regulators and Genomic Threats . Molecular Cell 73 : 398 – 411 OpenUrl CrossRef PubMed ↵ Crowley EA , Hermance NM , Herlihy CP , Manning AL ( 2022 ) Suppression of Chromosome Instability Limits Acquired Drug Resistance . Mol Cancer Ther 21 : 1583 – 1593 OpenUrl CrossRef PubMed ↵ Dunleavy EM , Almouzni G , Karpen GH ( 2011 ) H3. 3 is deposited at centromeres in S phase as a placeholder for newly assembled CENP-A in G1 phase . Nucleus 2 : 146 – 157 OpenUrl CrossRef PubMed Web of Science ↵ Earnshaw W , Bordwell B , Marino C , Rothfield N ( 1986 ) Three human chromosomal autoantigens are recognized by sera from patients with anti-centromere antibodies . The Journal of clinical investigation 77 : 426 – 430 OpenUrl CrossRef PubMed Web of Science ↵ Fachinetti D , Diego Folco H , Nechemia-Arbely Y , Valente LP , Nguyen K , Wong AJ , Zhu Q , Holland AJ , Desai A , Jansen LET et al. ( 2013 ) A two-step mechanism for epigenetic specification of centromere identity and function . Nature Cell Biology 15 : 1056 – 1066 OpenUrl CrossRef PubMed Web of Science ↵ Falk SJ , Guo LY , Sekulic N , Smoak EM , Mani T , Logsdon GA , Gupta K , Jansen LE , Van Duyne GD , Vinogradov SA et al. ( 2015 ) Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere . Science 348 : 699 – 703 OpenUrl Abstract / FREE Full Text ↵ Ferri F , Bouzinba-Segard H , Velasco G , Hube F , Francastel C ( 2009 ) Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase . Nucleic Acids Res 37 : 5071 – 5080 OpenUrl CrossRef PubMed ↵ Ganem NJ , Godinho SA , Pellman D ( 2009 ) A mechanism linking extra centrosomes to chromosomal instability . Nature 460 : 278 – 282 OpenUrl CrossRef PubMed Web of Science ↵ González-Loyola A , Fernández-Miranda G , Trakala M , Partida D , Samejima K , Ogawa H , Cañamero M , de Martino A , Martínez-Ramírez Á , de Cárcer G et al. ( 2015 ) Aurora B Overexpression Causes Aneuploidy and p21Cip1 Repression during Tumor Development . Mol Cell Biol 35 : 3566 – 3578 OpenUrl Abstract / FREE Full Text ↵ Gonzalo S , Blasco MA ( 2005 ) Role of Rb family in the epigenetic definition of chromatin . Cell Cycle 4 : 752 – 755 OpenUrl CrossRef PubMed Web of Science ↵ Gonzalo S , Garcia-Cao M , Fraga MF , Schotta G , Peters AH , Cotter SE , Eguia R , Dean DC , Esteller M , Jenuwein T et al. ( 2005 ) Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin . Nat Cell Biol 7 : 420 – 428 OpenUrl CrossRef PubMed Web of Science ↵ Guo LY , Allu PK , Zandarashvili L , McKinley KL , Sekulic N , Dawicki-McKenna JM , Fachinetti D , Logsdon GA , Jamiolkowski RM , Cleveland DW et al. ( 2017 ) Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition . Nat Commun 8 : 15775 OpenUrl CrossRef PubMed ↵ Hamperl S , Cimprich KA ( 2016 ) Conflict Resolution in the Genome: How Transcription and Replication Make It Work . Cell 167 : 1455 – 1467 OpenUrl CrossRef PubMed ↵ Hauf S , Cole RW , LaTerra S , Zimmer C , Schnapp G , Walter R , Heckel A , van Meel J , Rieder CL , Peters JM ( 2003 ) The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint . J Cell Biol 161 : 281 – 294 OpenUrl Abstract / FREE Full Text ↵ Herlihy CP , Hahn S , Hermance NM , Crowley EA , Manning AL ( 2021 ) Suv420 enrichment at the centromere limits Aurora B localization and function . J Cell Sci 134 ↵ Hernando E , Nahle Z , Juan G , Diaz-Rodriguez E , Alaminos M , Hemann M , Michel L , Mittal V , Gerald W , Benezra R et al. ( 2004 ) Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control . Nature 430 : 797 – 802 OpenUrl CrossRef PubMed Web of Science ↵ Huang H , Lampson M , Efimov A , Yen TJ ( 2018 ) Chromosome instability in tumor cells due to defects in Aurora B mediated error correction at kinetochores . Cell Cycle 17 : 2622 – 2636 OpenUrl CrossRef PubMed ↵ Ideue T , Cho Y , Nishimura K , Tani T ( 2014 ) Involvement of satellite I noncoding RNA in regulation of chromosome segregation . Genes Cells 19 : 528 – 538 OpenUrl CrossRef PubMed ↵ Isaac CE , Francis SM , Martens AL , Julian LM , Seifried LA , Erdmann N , Binné UK , Harrington L , Sicinski P , Bérubé NG et al. ( 2006 ) The retinoblastoma protein regulates pericentric heterochromatin . Mol Cell Biol 26 : 3659 – 3671 OpenUrl Abstract / FREE Full Text ↵ Ishak CA , Marshall AE , Passos DT , White CR , Kim SJ , Cecchini MJ , Ferwati S , MacDonald WA , Howlett CJ , Welch ID ( 2016 ) An RB-EZH2 complex mediates silencing of repetitive DNA sequences . Molecular cell 64 : 1074 – 1087 OpenUrl CrossRef PubMed ↵ Jambhekar A , Emerman AB , Schweidenback CT , Blower MD ( 2014 ) RNA stimulates Aurora B kinase activity during mitosis . PLoS One 9 : e100748 OpenUrl CrossRef PubMed ↵ Jansen LE , Black BE , Foltz DR , Cleveland DW ( 2007 ) Propagation of centromeric chromatin requires exit from mitosis . The Journal of cell biology 176 : 795 – 805 OpenUrl Abstract / FREE Full Text ↵ Jeppesen P , Mitchell A , Turner B , Perry P ( 1992 ) Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes . Chromosoma 101 : 322 – 332 OpenUrl CrossRef PubMed Web of Science ↵ Kabeche L , Nguyen HD , Buisson R , Zou L ( 2018 ) A mitosis-specific and R loop–driven ATR pathway promotes faithful chromosome segregation . Science 359 : 108 – 114 OpenUrl Abstract / FREE Full Text ↵ Keohane AM , O’Neill L P , Belyaev ND , Lavender JS , Turner BM ( 1996 ) X-Inactivation and histone H4 acetylation in embryonic stem cells . Dev Biol 180 : 618 – 630 OpenUrl CrossRef PubMed Web of Science ↵ Kleyman M , Kabeche L , Compton DA ( 2014 ) STAG2 promotes error correction in mitosis by regulating kinetochore-microtubule attachments . J Cell Sci 127 : 4225 – 4233 OpenUrl Abstract / FREE Full Text ↵ Knowlton AL , Lan W , Stukenberg PT ( 2006 ) Aurora B is enriched at merotelic attachment sites, where it regulates MCAK . Curr Biol 16 : 1705 – 1710 OpenUrl CrossRef PubMed Web of Science ↵ Kourmouli N , Sun Y-m , van der Sar S , Singh PB , Brown JP ( 2005 ) Epigenetic regulation of mammalian pericentric heterochromatin in vivo by HP1 . Biochemical and biophysical research communications 337 : 901 – 907 OpenUrl CrossRef PubMed Web of Science ↵ Liu H , Qu Q , Warrington R , Rice A , Cheng N , Yu H ( 2015 ) Mitotic Transcription Installs Sgo1 at Centromeres to Coordinate Chromosome Segregation . Mol Cell 59 : 426 – 436 OpenUrl CrossRef PubMed ↵ Lockhart A , Pires VB , Bento F , Kellner V , Luke-Glaser S , Yakoub G , Ulrich HD , Luke B ( 2019 ) RNase H1 and H2 Are Differentially Regulated to Process RNA-DNA Hybrids . Cell Rep 29 : 2890 – 2900 .e2895 OpenUrl CrossRef PubMed ↵ Manning AL , Dyson NJ ( 2011 ) pRB, a tumor suppressor with a stabilizing presence . Trends in Cell Biology 21 : 433 – 441 OpenUrl CrossRef PubMed Web of Science ↵ Manning AL , Longworth MS , Dyson NJ ( 2010 ) Loss of pRB causes centromere dysfunction and chromosomal instability . Genes Dev 24 : 1364 – 1376 OpenUrl Abstract / FREE Full Text ↵ Manning AL , Yazinski SA , Nicolay B , Bryll A , Zou L , Dyson NJ ( 2014 ) Suppression of genome instability in pRB-deficient cells by enhancement of chromosome cohesion . Mol Cell 53 : 993 – 1004 OpenUrl CrossRef PubMed ↵ Martins NM , Bergmann JH , Shono N , Kimura H , Larionov V , Masumoto H , Earnshaw WC ( 2016 ) Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3 . Mol Biol Cell 27 : 177 – 196 OpenUrl Abstract / FREE Full Text ↵ Martins NMC , Cisneros-Soberanis F , Pesenti E , Kochanova NY , Shang WH , Hori T , Nagase T , Kimura H , Larionov V , Masumoto H et al. ( 2020 ) H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory . J Cell Sci 133 ↵ Matos DA , Zhang J-M , Ouyang J , Nguyen HD , Genois M-M , Zou L ( 2020 ) ATR Protects the Genome against R Loops through a MUS81-Triggered Feedback Loop . Molecular Cell 77 : 514 – 527 .e514 OpenUrl CrossRef PubMed ↵ McNulty SM , Sullivan LL , Sullivan BA ( 2017 ) Human Centromeres Produce Chromosome-Specific and Array-Specific Alpha Satellite Transcripts that Are Complexed with CENP-A and CENP-C . Dev Cell 42 : 226 – 240 .e226 OpenUrl CrossRef PubMed ↵ Melters DP , Bradnam KR , Young HA , Telis N , May MR , Ruby JG , Sebra R , Peluso P , Eid J , Rank D ( 2013 ) Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution . Genome biology 14 : 1 – 20 OpenUrl CrossRef ↵ Muñoz-Barrera M , Monje-Casas F ( 2014 ) Increased Aurora B activity causes continuous disruption of kinetochore-microtubule attachments and spindle instability . Proc Natl Acad Sci U S A 111 : E3996 – 4005 OpenUrl Abstract / FREE Full Text ↵ Musacchio A , Salmon ED ( 2007 ) The spindle-assembly checkpoint in space and time . Nat Rev Mol Cell Biol 8 : 379 – 393 OpenUrl CrossRef PubMed Web of Science ↵ Nicolay BN , Danielian PS , Kottakis F , Lapek JD , Sanidas I , Miles WO , Dehnad M , Tschöp K , Gierut JJ , Manning AL et al. ( 2015 ) Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation . Genes & Development 29 : 1875 – 1889 OpenUrl Abstract / FREE Full Text ↵ Nielsen SJ , Schneider R , Bauer U-M , Bannister AJ , Morrison A , O’Carroll D , Firestein R , Cleary M , Jenuwein T , Herrera RE ( 2001 ) Rb targets histone H3 methylation and HP1 to promoters . Nature 412 : 561 – 565 OpenUrl CrossRef PubMed Web of Science ↵ Palmer DK , O’Day K , Trong H , Charbonneau H , Margolis RL ( 1991 ) Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone . Proceedings of the National Academy of Sciences 88 : 3734 – 3738 OpenUrl Abstract / FREE Full Text ↵ Perea-Resa C , Blower MD ( 2018 ) Centromere Biology: Transcription Goes on Stage . Mol Cell Biol 38 ↵ Perea-Resa C , Bury L , Cheeseman IM , Blower MD ( 2020 ) Cohesin Removal Reprograms Gene Expression upon Mitotic Entry . Mol Cell 78 : 127 – 140 .e127 OpenUrl CrossRef PubMed ↵ Petermann E , Lan L , Zou L ( 2022 ) Sources, resolution and physiological relevance of R-loops and RNA-DNA hybrids . Nat Rev Mol Cell Biol 23 : 521 – 540 OpenUrl CrossRef PubMed ↵ Peters AH , O’Carroll D , Scherthan H , Mechtler K , Sauer S , Schöfer C , Weipoltshammer K , Pagani M , Lachner M , Kohlmaier A ( 2001 ) Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability . Cell 107 : 323 – 337 OpenUrl CrossRef PubMed Web of Science ↵ Politi V , Perini G , Trazzi S , Pliss A , Raska I , Earnshaw WC , Della Valle G ( 2002 ) CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains . J Cell Sci 115 : 2317 – 2327 OpenUrl Abstract / FREE Full Text ↵ Quénet D , Dalal Y ( 2014 ) A long non-coding RNA is required for targeting centromeric protein A to the human centromere . eLife 3 : e26016 OpenUrl CrossRef PubMed ↵ Rea S , Eisenhaber F , O’Carroll D , Strahl BD , Sun Z-W , Schmid M , Opravil S , Mechtler K , Ponting CP , Allis CD ( 2000 ) Regulation of chromatin structure by site-specific histone H3 methyltransferases . Nature 406 : 593 – 599 OpenUrl CrossRef PubMed Web of Science ↵ Rice JC , Briggs SD , Ueberheide B , Barber CM , Shabanowitz J , Hunt DF , Shinkai Y , Allis CD ( 2003 ) Histone Methyltransferases Direct Different Degrees of Methylation to Define Distinct Chromatin Domains . Molecular Cell 12 : 1591 – 1598 OpenUrl CrossRef PubMed Web of Science ↵ Rogakou EP , Pilch DR , Orr AH , Ivanova VS , Bonner WM ( 1998 ) DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139* . Journal of Biological Chemistry 273 : 5858 – 5868 OpenUrl Abstract / FREE Full Text ↵ Rogers SG , Weiss B ( 1980 ) [26] Exonuclease III of Escherichia coli K-12, an AP endonuclease . In: Methods in Enzymology , pp. 201 – 211 . Academic Press : ↵ Rošić S , Erhardt S ( 2016 ) No longer a nuisance: long non-coding RNAs join CENP-A in epigenetic centromere regulation . Cellular and Molecular Life Sciences 73 : 1387 – 1398 OpenUrl CrossRef PubMed ↵ Rošić S , Köhler F , Erhardt S ( 2014 ) Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division . J Cell Biol 207 : 335 – 349 OpenUrl Abstract / FREE Full Text ↵ Saayman X , Graham E , Leung CWB , Esashi F ( 2023a ) exo-FISH: Protocol for detecting DNA breaks in repetitive regions of mammalian genomes . STAR Protoc 4 : 102487 OpenUrl CrossRef PubMed ↵ Saayman X , Graham E , Nathan WJ , Nussenzweig A , Esashi F ( 2023b ) Centromeres as universal hotspots of DNA breakage, driving RAD51-mediated recombination during quiescence . Molecular Cell 83 : 523 – 538 .e527 OpenUrl CrossRef PubMed ↵ Sanidas I , Morris R , Fella KA , Rumde PH , Boukhali M , Tai EC , Ting DT , Lawrence MS , Haas W , Dyson NJ ( 2019 ) A Code of Mono-phosphorylation Modulates the Function of RB . Mol Cell 73 : 985 – 1000 .e1006 OpenUrl CrossRef PubMed ↵ Santos-Pereira JM , Aguilera A ( 2015 ) R loops: new modulators of genome dynamics and function . Nat Rev Genet 16 : 583 – 597 OpenUrl CrossRef PubMed ↵ Schotta G , Lachner M , Sarma K , Ebert A , Sengupta R , Reuter G , Reinberg D , Jenuwein T ( 2004 ) A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin . Genes Dev 18 : 1251 – 1262 OpenUrl Abstract / FREE Full Text ↵ Sedelnikova OA , Rogakou EP , Panyutin IG , Bonner WM ( 2002 ) Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody . Radiat Res 158 : 486 – 492 OpenUrl CrossRef PubMed Web of Science ↵ Siddiqui H , Fox SR , Gunawardena RW , Knudsen ES ( 2007 ) Loss of RB compromises specific heterochromatin modifications and modulates HP1alpha dynamics . J Cell Physiol 211 : 131 – 137 OpenUrl CrossRef PubMed ↵ Sidhwani P , Straight AF ( 2023 ) Epigenetic inheritance and boundary maintenance at human centromeres . Curr Opin Struct Biol 82 : 102694 OpenUrl CrossRef PubMed ↵ Sullivan BA , Karpen GH ( 2004 ) Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin . Nat Struct Mol Biol 11 : 1076 – 1083 OpenUrl CrossRef PubMed Web of Science ↵ Talbert PB , Kasinathan S , Henikoff S ( 2018 ) Simple and Complex Centromeric Satellites in Drosophila Sibling Species . Genetics 208 : 977 – 990 OpenUrl Abstract / FREE Full Text ↵ Thomas M , White RL , Davis RW ( 1976 ) Hybridization of RNA to double-stranded DNA: formation of R-loops . Proc Natl Acad Sci U S A 73 : 2294 – 2298 OpenUrl Abstract / FREE Full Text ↵ Ting DT , Lipson D , Paul S , Brannigan BW , Akhavanfard S , Coffman EJ , Contino G , Deshpande V , Iafrate AJ , Letovsky S et al. ( 2011 ) Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers . Science 331 : 593 – 596 OpenUrl Abstract / FREE Full Text ↵ Vandel L , Nicolas E , Vaute O , Ferreira R , Ait-Si-Ali S , Trouche D ( 2001 ) Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase . Molecular and Cellular Biology 21 : 6484 – 6494 OpenUrl Abstract / FREE Full Text ↵ Watanabe R , Hara M , Okumura E-i , Hervé S , Fachinetti D , Ariyoshi M , Fukagawa T ( 2019 ) CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization . Journal of Cell Biology 218 : 4042 – 4062 OpenUrl Abstract / FREE Full Text ↵ Wong LH , Brettingham-Moore KH , Chan L , Quach JM , Anderson MA , Northrop EL , Hannan R , Saffery R , Shaw ML , Williams E et al. ( 2007 ) Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere . Genome Res 17 : 1146 – 1160 OpenUrl Abstract / FREE Full Text ↵ Zamalloa LG , Pruitt MM , Hermance NM , Gali H , Flynn RL , Manning AL ( 2023 ) RB loss sensitizes cells to replication-associated DNA damage after PARP inhibition by trapping . Life Sci Alliance 6 ↵ Zhu Q , Hoong N , Aslanian A , Hara T , Benner C , Heinz S , Miga KH , Ke E , Verma S , Soroczynski J et al. ( 2018 ) Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer . Mol Cell 70 : 842 – 853 .e847 OpenUrl CrossRef PubMed ↵ Zhu Q , Pao GM , Huynh AM , Suh H , Tonnu N , Nederlof PM , Gage FH , Verma IM ( 2011 ) BRCA1 tumour suppression occurs via heterochromatin-mediated silencing . Nature 477 : 179 – 184 OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted June 05, 2025. Download PDF 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 RB dependent transcriptional regulation at mitotic centromeres preserves genome stability 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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Share RB dependent transcriptional regulation at mitotic centromeres preserves genome stability Elizabeth A Crowley , Amity L Manning bioRxiv 2025.06.04.657825; doi: https://doi.org/10.1101/2025.06.04.657825 Share This Article: Copy Citation Tools RB dependent transcriptional regulation at mitotic centromeres preserves genome stability Elizabeth A Crowley , Amity L Manning bioRxiv 2025.06.04.657825; doi: https://doi.org/10.1101/2025.06.04.657825 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42005) Biophysics (21472) Cancer Biology (18624) Cell Biology (25534) Clinical Trials (138) Developmental Biology (13391) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40437) Molecular Biology (17200) Neuroscience (88697) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4829) Physiology (7653) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9827) Zoology (2272)