Defining the DNA Binding Specificity of GRHL2

demonstrate that combining SNAP array measurements with genome-wide ChIP-seq studies ĞŶĂďůĞƐĨƵŶĐƟŽŶĂůƐĞƉĂƌĂƟŽŶŽĨĚŝƌĞĐƚƐĞƋƵĞŶĐĞ-encoded GRHL2 binding from cofactor- ĚĞƉĞŶĚĞŶƚŽĐĐƵƉĂŶĐLJ͕ƌĞǀĞĂůŝŶŐĚŝƐƟŶĐƚĐůĂƐƐĞƐŽĨ'Z,>Ϯ-associated genomic loci. A dense cluster oĨ GRHL2 moƟĨs corresƉonds to a ŚigŚly occuƉied CŚIP-seq region /Ŷ ĞdžĂŵŝŶŝŶŐ ŚŽǁ ŽŌĞŶ ƚŚĞ 'Z,>Ϯ ďŝŶĚŝŶŐ ŵŽƟĨ ŽĐĐƵƌƐ ĂĐƌŽƐƐ ƚŚĞŐĞŶŽŵĞ͕ Ălow- ĐŽŵƉůĞdžŝƚLJregion on chromosome 7 that contains an unusually high density of GRHL2 consensus ŵŽƟĨƐ ǁĂƐ ŝĚĞŶƟĮĞĚ ;&ŝŐƵƌĞ ϳͿ͘ tŝƚŚŝŶ ƚŚŝƐ ŝŶƚĞƌǀĂů ĨƌŽŵ ĐŚƌϳ͗ϭϱϱ͕ϭϮϰ͕ϬϬϰ-155,124,759, ŵƵůƟƉůĞ ĞdžĂĐƚ ĐĂŶŽŶŝĐĂů ''dd ŵŽƟĨƐ ǁĞƌĞ ƉƌĞƐĞŶƚ͕ ŝŶƚĞƌƐƉĞƌƐĞĚ ǁŝƚŚ ĂĚĚŝƟŽŶĂů ƐŝŶŐůĞ- .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 21 mismatch variants. This arrangement created a concentrated cluster of closely spaced GRHL2 ƌĞĐŽŐŶŝƟŽŶƐŝƚĞƐĂĐƌŽƐƐĂƌĞůĂƟǀĞůLJƐŚŽƌƚƐƚƌĞƚĐŚŽĨE͘Ś/W-seq data from our group (7, 30) showed strong GRHL2 enrichment at the edges of this region under both datasets (Figure 7B, ƚŽƉͿ͘tŚĞŶŵĂƉƉĞĚŽŶƚŚĞ^EWĂƌƌĂLJ͕ƉƌŽďĞƐƟůĞĚĞǀĞƌLJƐŝdžďĂƐĞƉĂŝƌƐĂĐƌŽƐƐƚŚĞƐĂŵĞŝŶƚĞƌǀĂů produced a broad band of elevated binding signal within the central region ;&ŝŐƵƌĞϳ͕ďŽƩŽŵͿ͘ ZĂƚŚĞƌƚŚĂŶĂƐŝŶŐůĞĚŝƐĐƌĞƚĞďŝŶĚŝŶŐƉĞĂŬ͕ƚŚĞ^EWƐŝŐŶĂůŇƵĐƚƵĂƚĞĚĂĐƌŽƐƐƚŚĞƟůĞĚƉƌŽďĞƐ͕ ǁŚŝĐŚŝƐĐŽŶƐŝƐƚĞŶƚǁŝƚŚŵƵůƟƉůĞďŝŶĚŝŶŐĞǀĞŶƚƐĂƌŝƐŝŶŐĨƌŽŵƚŚĞƌĞƉĞĂƚĞĚĐŽŶƐĞŶƐƵƐĂŶĚŶĞĂƌ- ĐŽŶƐĞŶƐƵƐ ŵŽƟĨƐ͘ dŚĞ ŽďƐĞƌǀĞĚ ĚŝīĞƌĞnce between the ChIP-seq and SNAP data could be ĞdžƉůĂŝŶĞĚďLJŝŵƉƌĞĐŝƐĞŵĂƉƉŝŶŐŽĨŚ/W-seq reads in this low-ĐŽŵƉůĞdžŝƚLJƌĞŐŝŽŶƚŚĂƚƌĞƐƵůƚed in ƚǁŽƉĞĂŬƐŽŶĞŝƚŚĞƌƐŝĚĞŽĨƚŚĞƌĞŐŝŽŶ͘/ŶƉĂƌƟĐƵůĂƌ͕ƚŚĞŚ/W-seq peak on one side of the region may represent the perfect GRHL2 consensus (5’-AACCGGTT-3’) reads while the ChIP-seq peak on other side of the region may represent the primary mismatch site (5’-AAACGGTT-3’) reads. ůƚĞƌŶĂƟǀĞůLJ͕ƚŚŝƐƌĞŐŝŽŶŵĂLJďĞĐŚƌŽŵĂƟŶŝŶĂĐĐĞƐƐŝďůĞƚŽƚŚĞŚ/W-ƐĞƋĂŶƟďŽĚLJƌĞĂŐĞŶƚƐ͘These ŽďƐĞƌǀĂƟŽŶƐŝŶĚŝĐĂƚĞƚŚĂƚƚŚŝƐƌĞŐŝŽŶƉŽƚĞŶƟĂůůLJrepresents a highly occupied GRHL2 locus driven ďLJƚŚĞƉƌĞƐĞŶĐĞŽĨŵĂŶLJĐůŽƐĞůLJƐƉĂĐĞĚďŝŶĚŝŶŐƐŝƚĞƐ͘dŚĞĐŽŵďŝŶĂƟŽŶŽĨĚĞŶƐĞĐŽŶƐĞŶƐƵƐŵŽƟĨƐ and favorable single-mismatch variants likely creates a local DNA environment that strongly favors 'Z,>ϮďŝŶĚŝŶŐĂĐƌŽƐƐƚŚĞĞŶƟƌĞŝŶƚĞƌǀĂů͘  /^h^^/KE In this study, we used a customized high-density genomic SNAP DNA-binding array to biochemically ĚĞĮŶĞ͕ Ăƚ Ɛŝdž-ďĂƐĞ ƌĞƐŽůƵƟŽŶ͕ ƚŚĞ ƐĞƋƵĞŶĐĞ ĨĞĂƚƵƌĞƐ ƚŚĂƚ ŐŽǀĞƌŶ 'Z,>Ϯ E ƌĞĐŽŐŶŝƟŽŶ ĂĐƌŽƐƐ ƚŚĞ ŐĞŶŽŵĞ͘ LJ ŝŶƚĞŐƌĂƟŶŐ ĚŝƌĞĐƚ ďŝŶĚŝŶŐ ŵĞĂƐƵƌĞŵĞŶƚƐ ǁŝƚŚ ƐƚƌƵĐƚƵƌĂů .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 22 ŝŶƚĞƌƉƌĞƚĂƟŽŶĂŶĚŚ/W-ƐĞƋĚĂƚĂ͕ǁĞĚŝƐƟŶŐƵŝƐŚŵŽƟĨ-encoded GRHL2 direct binding events from cofactor-dependent occupancy to ƌĞƐŽůǀĞŚŽǁŵŽƟĨĐŽŵƉŽƐŝƟŽŶ͕ŇĂŶŬŝŶŐƐĞƋƵĞŶĐĞ͕ĂŶĚŵŽƟĨ ƐƉĂĐŝŶŐĐŽůůĞĐƟǀĞůLJdictate GRHL2 binding ĐŚĂƌĂĐƚĞƌŝƐƟĐƐ. While prior studies have established GRHL2 as a co-ƌĞŐƵůĂƚŽƌŽĨƚƌĂŶƐĐƌŝƉƟŽŶƚŚƌŽƵŐŚŝŶƚĞƌĂĐƟŽŶƐǁŝƚŚĨĂĐƚŽƌƐƐƵĐŚĂƐZɲand PR and ƚŚƌŽƵŐŚŵŽĚƵůĂƟŽŶŽĨĐŚƌŽŵĂƟŶĂĐĐĞƐƐŝďŝůŝƚLJ͕ƚŚĞƐĞĂƉƉƌŽĂĐŚĞƐĚŽŶŽƚĚŝīĞƌĞŶƟĂƚĞ direct DNA binding from indirect, cofactor-mediated recruitm ent. The present work addresses this gap by ĚĞĮŶŝŶŐƚŚĞŝŶƚƌŝŶƐŝĐƐĞƋƵĞŶĐĞĚĞƚĞƌŵŝŶĂŶƚƐŽĨ'Z,>ϮďŝŶĚŝŶŐŝŶĚĞƉĞŶĚĞŶƚŽĨĐŚƌŽŵĂƟŶĐŽŶƚĞdžƚ (50, 51). Across more than 772,732 genomic probe sequences, GRHL2 binding prefers a conserved     ' ' d d ŵ Ž Ɵ Ĩ ͘ ^ Ƶ ď Ɛ Ɵ ƚ Ƶ Ɵ Ž Ŷ Ɛ Ă ƚ Ɖ Ž Ɛ ŝ Ɵons 3 (C) ĂŶĚ ƉŽƐŝƟŽŶ ϲ (G) produced the steepest ƌĞĚƵĐƟŽŶƐŝŶ^EWŝŶƚĞŶƐŝƚLJ. SƵďƐƟƚƵƟŽŶƐĂƚƚŚĞcentral ';ƉŽƐŝƟŽŶƐϰ-5) had minor ĞīĞĐƚƐ, and ƐƵďƐƟƚƵƟŽŶƐĂƚƚŚĞŽƵƚĞƌͬdƉŽƐŝƟŽŶƐǁĞƌĞŵŽƌĞƚŽůĞƌĂƚĞĚ͘dŚĞƐĞƌĞƐƵůƚƐĂƌĞ consistent with ƐƚƌƵĐƚƵƌĂůƐƚƵĚŝĞƐŽĨƚŚĞ'ƌĂŝŶLJŚĞĂĚͬWϮĨĂŵŝůLJ(43). TŚĞƌĞĐŽŐŶŝƟŽŶŚĞůŝdžŽĨƚŚĞǁŝŶŐĞĚ-ŚĞůŝdž DNA-ďŝŶĚŝŶŐ ĚŽŵĂŝŶ ĐŽŶƚĂĐƚƐ ƚŚĞ ƉŽƐŝƟŽŶƐ ϯ ĂŶĚ ϲ ŽĨ ƚŚĞ ''dd ŵŽƟĨ ǁŝƚŚ ŶŽ ĚŝƌĞĐƚ contacts to ƚŚĞĐĞŶƚƌĂů'͕ƌĞƐƵůƟŶŐŝŶ a dždž'ĐŽƌĞ͘The limited backbone contacts outside of the octamer ĐŽƌĞ ŵŽƟĨfound in the crystal structure (43) Ɛ Ƶ Ő Ő Ğ Ɛ ƚ ƚ Ś Ă ƚ ƚ Ś Ğ Ň Ă Ŷ Ŭ ŝ Ŷ Ő Ɖ ƌ Ğ Ĩ Ğ ƌ Ğ Ŷ Đ Ğ Ɛ  uncovered in our analysis may be driven by DNA structural features, such as major groove ŐĞŽŵĞƚƌLJ ŝŶŇƵĞŶĐĞĚ ďLJ ͬd ĐŽŶƚĞŶƚ͘ The SSL and mismatch analyses also ĞdžŚŝďŝƚĞĚ this enrichment of tolerated single-ďĂƐĞƐƵďƐƟƚƵƟŽŶƐĂƚƚŚĞŵŝĚĚůĞ'ƉŽƐŝƟŽŶƐŽĨƚŚĞŵŽƟĨ͕ǁŝƚŚĂ ƐƚƌŽŶŐ ďŝĂƐ ƚŽǁĂƌĚ їdͬ ĂŶĚ 'їͬd ĐŚĂŶŐĞƐ͘ KŶĞ ƉŽƐƐŝďůĞ ĞdžƉůĂŶĂƟŽŶ ŝƐ ƚŚĂƚ ƚŚĞƐĞ ƐƵďƐƟƚƵƟŽŶƐ ƉƌĞƐĞƌǀĞ E ƐŚĂƉĞ Žƌ ŚLJĚƌŽŐĞŶ-bonding geometry in a way that ŝƐ ƉĂƌƟĂůůLJ .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 23 ĐŽŵƉĂƟďůĞǁŝƚŚ'Z,>ϮƌĞĐŽŐŶŝƟŽŶ͕ĂůůŽǁŝŶŐůŽǁ-ĂĸŶŝƚLJďŝŶĚŝŶŐƌĂƚŚĞƌƚŚĂŶƚŚĞĐŽŵƉůĞƚĞůŽƐƐ ŽĨƚŚĞŝŶƚĞƌĂĐƟŽŶ͘ The SSL analysis provides ƋƵĂŶƟƚĂƟǀĞdetail towards a structural model in which the central dždž'ĂĐƚƐĂƐƚŚĞƉƌŝŶĐŝƉĂůĞŶĞƌŐĞƟĐcontributor for GRHL2-EƌĞĐŽŐŶŝƟŽŶ͕ǁŚŝůĞŇĂŶŬŝŶŐ ƐĞƋƵĞŶĐĞƐĂŶĚƚŚĞĐĞŶƚƌĂů'ƚƵŶĞďŝŶĚŝŶŐĂĸŶŝƚLJand ƐƉĞĐŝĮĐŝƚLJ. TŚŝƐůĞǀĞůŽĨƌĞƐŽůƵƟŽŶĞdžƚĞŶĚƐ ďĞLJŽŶĚ ƚƌĂĚŝƟŽŶĂů ŵŽƟĨ ĞŶƌŝĐŚŵĞŶƚ ĂŶĂůLJƐĞƐ o f C h I P - s e q p e a k s ďLJ ƉƌŽǀŝĚŝŶŐ Ă ƋƵĂŶƟƚĂƟǀĞ ĚĞƐĐƌŝƉƟŽŶŽĨĞdžĂĐƚůLJǁŚĞƌĞ'Z,>ϮŵĂLJďŝŶĚǁŝƚŚŝŶa genomic region to a “sub-peak” level. This ĞŶĂďůĞƐ ĚŝƌĞĐƚ ŝŶƚĞƌƉƌĞƚĂƟŽŶ ŽĨ ŐĞŶŽŵŝĐ 'Z,>Ϯ ŽĐĐƵƉĂŶĐLJto ĚŝƐƟŶŐƵŝƐh sequence-driven ďŝŶĚŝŶŐĨƌŽŵŝŶĚŝƌĞĐƚƌĞĐƌƵŝƚŵĞŶƚŽďƐĞƌǀĞĚŝŶĐŚƌŽŵĂƟŶ-based assays. dŚĞĞŶƌŝĐŚŵĞŶƚŽĨƚŽůĞƌĂƚĞĚƐƵďƐƟƚƵƟŽŶƐĂƚƚŚĞĐĞŶƚƌĂů'ƉŽƐŝƟŽŶƐĨƵƌƚŚĞƌƐƵŐŐĞƐƚƐƚŚĂƚ 'Z,>ϮďŝŶĚŝŶŐŵĂLJďĞƌĞůĂƟǀĞůLJƌŽďƵƐƚƚŽƐĞƋƵĞŶĐĞǀĂƌŝĂƟŽŶĂƚƉ'ĚŝŶƵĐůĞŽƟĚĞƐ͕ǁŚŝĐŚĂƌĞ known to be highly mutable in mamma lian genomes due to cytosine deamina ƟŽŶ (13). The ŽďƐĞƌǀĞĚƚŽůĞƌĂŶĐĞƚŽƐƉĞĐŝĮĐїdͬĂŶĚ'їͬdƐƵďƐƟƚƵƟŽŶƐbrings forth the idea that GRHL2 ďŝŶĚŝŶŐĐĂŶƉĞƌƐŝƐƚ͕ĂƚƌĞĚƵĐĞĚĂĸŶŝƚLJ͕ŝŶƌĞŐƵůĂƚŽƌLJƌĞŐŝŽŶƐƵŶĚĞƌŐŽŝŶŐƉ'ĞƌŽƐŝŽŶ͘dŚŝƐŵĂLJ ŚĂǀĞŝŵƉůŝĐĂƟŽŶƐĨŽƌƚŚĞĞǀŽůƵƟŽŶŽĨ'Z,>ϮďŝŶĚŝŶŐƐŝƚĞƐĂŶĚĨŽƌƚŚĞŵĂŝŶƚĞŶĂŶĐĞŽĨƌĞŐƵůĂƚŽƌLJ ĨƵŶĐƟŽŶŝŶŐĞŶŽŵŝĐƌĞŐŝŽŶƐ ƚŚĂƚĂƌĞŬŶŽǁŶƚŽďĞŵƵƚĂƟŽŶĂůůLJĂĐƟǀĞ. KƵƌƐƉĂĐŝŶŐĂŶĚŽƌŝĞŶƚĂƟŽŶĂŶĂůLJƐĞƐĚĞŵŽŶƐƚƌĂƚĞƚŚĂƚ'Z,>ϮďŝŶĚŝŶŐŝƐĂīĞĐƚĞĚ ďLJŵŽƟĨ ŽƌŐ ĂŶŝnjĂƟŽŶ͘W ĂŝƌĞĚŵŽƟĨƐŝŶĂĨ ŽƌǁĂƌĚ–ĨŽƌǁĂƌĚ ŽƌŝĞŶƚĂƟŽŶ(AACCGGTT-AACCGGTT) ĞdžŚŝďŝƚĞĚ ƚŚĞƐƚƌŽŶŐĞƐƚďŝŶĚŝŶŐĂƚƐŚŽƌƚĚŝƐƚĂŶĐĞƐĂŶĚĚŝƐƉůĂLJĞĚĂƉĞƌŝŽĚŝĐƉĂƩĞƌŶǁŝƚŚĂŶĂƉƉƌŽdžŝŵĂƚĞ 5.5-bp interval, consistent with half of the DNA he lical turn (38). This periodicity suggests that ŽƉƟŵĂů ďŝŶĚŝŶŐ ŽĐĐƵƌƐ ǁŚĞŶ ŵŽƟĨƐ ĂƌĞ ƉŽƐŝƟŽŶĞĚ ŽŶ ƚŚĞ ƐĂŵĞ ĨĂĐĞ ŽĨ ƚŚĞ E ŚĞůŝdž͕which supports a ŵŽĚĞůŽĨĐŽŽƉĞƌĂƟǀĞŽƌĚŝŵĞƌ binding. While previous studies have suggested that .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 24 ŵĞŵďĞƌƐ ŽĨ ƚŚĞ 'ƌĂŝŶLJŚĞĂĚ ĨĂŵŝůLJ ĐĂŶ ĨŽƌŵ ĚŝŵĞƌƐ͕ ƚŚĞ ƉƌĞƐĞŶƚ ƌĞƐƵůƚƐ ĚĞĮŶĞ ƚŚĞ ƐƉĂƟĂů constraints of ƚŚĞƐĞ ŝŶƚĞƌĂĐƟŽŶƐ ĂŶĚ ƉƌŽǀŝĚĞ Ă ƐƚƌƵĐƚƵƌĂů ďĂƐŝƐ ĨŽƌ ŵŽƟĨ ĐůƵƐƚĞƌŝŶŐ ŽďƐĞƌǀĞĚ within GRHL2-bound regulatory regions. dŚĞ ĚĞŶƐĞ ŵŽƟĨ ĐůƵƐƚĞƌ ŽŶ ĐŚƌŽŵŽƐŽŵĞ ϳ͕ ǁŚĞƌĞ repeated consensus and near-ĐŽŶƐĞŶƐƵƐŵŽƟĨƐƉƌŽĚƵĐĞĚĂďƌŽĂĚƌĞŐŝŽŶŽĨĞůĞǀĂƚĞĚ^EWƐŝŐŶĂů and ChIP-ƐĞƋŽĐĐƵƉĂŶĐLJ͕ƉƌŽǀŝĚĞƐĂŶĂƚƵƌĂůŐĞŶŽŵŝĐĞdžĂŵƉůĞŽĨŚŽǁƐƵĐŚŵƵůƟǀĂůĞŶƚďŝŶĚŝŶŐ environments may occur in the genome. dŚĞƐĞ ĮŶĚŝŶŐƐ ƉƌŽǀŝĚĞ Ămethod ĨŽƌ ŝŶƚĞƌƉƌĞƟŶŐ 'Z,>Ϯ ĐŽ-occupancy with other ƚƌĂŶƐĐƌŝƉƟŽŶ ĨĂĐƚŽƌƐ ŝŶ ŐĞŶŽŵŝĐ ƐƚƵĚŝĞƐ͘Recent work has demonstrated that GRHL2 co-binds ĞŶŚĂŶĐĞƌ ƌĞŐŝŽŶƐ ǁŝƚŚ WZ ĂŶĚ ĐŽŶƚƌŝďƵƚĞƐ ƚŽ ĐŚƌŽŵĂƟŶ ĂĐĐĞƐƐŝďŝůŝƚLJ ĂŶĚ ƚƌĂŶƐĐƌŝƉƟŽŶĂů ƌĞŐƵůĂƟŽŶ ŝŶ ŚŽƌŵŽŶĞ-responsive breast cancer cells (50). However, such approaches cannot resolve whether GRHL2 is directly engaging DNA at these sites or is recruited through protein- ƉƌŽƚĞŝŶŝŶƚĞƌĂĐƟŽŶƐ͘LJĚŝƐƟŶŐƵŝƐŚŝŶŐĚŝƌĞĐƚŵŽƟĨ-driven binding from indirect occupancy, our

act in complement with genome-wide studies to show that only a subset of GRHL2 ChIP- seq peaks represent sequence- ĞŶĐŽĚĞĚ ďŝŶĚŝŶŐ ĞǀĞŶƚƐ͕ ǁŚŝůĞ ŽƚŚĞƌƐ ŵĂLJ ƌĞŇĞĐƚ ĐŽĨĂĐƚŽƌ- ŵĞĚŝĂƚĞĚ ƌĞĐƌƵŝƚŵĞŶƚ͘ dŚŝƐ ĚŝƐƟŶĐƟŽŶ ŚĂƐ ŝŵƉŽƌƚĂŶƚ ŝŵƉůŝĐĂƟŽŶƐĨŽƌ ŝŶƚĞƌƉƌĞƟŶŐ ŐĞŶŽŵŝĐ occupancy data anĚĨŽƌŝĚĞŶƟĨLJŝŶŐĨƵŶĐƟŽŶĂůůLJƌĞůĞǀĂŶƚ'Z,>ϮďŝŶĚŝŶŐƐŝƚĞƐ͘ dŚŝƐŽďƐĞƌǀĂƟŽŶŝƐ consistent with prior transcriptomic and cistromic studies showing that GRHL2 interacts with ERɲ͕ FŽdžϭ͕ĂŶĚŽƚŚĞƌƚƌĂŶƐĐƌŝƉƟŽŶĂůƌĞŐƵůĂƚŽƌƐĂŶĚĨƌĞƋƵĞŶƚůLJƉĂƌƟĐŝƉĂƚĞƐŝŶĞŶŚĂŶĐĞƌĞŶǀŝƌŽŶŵĞŶƚƐ ƌĂƚŚĞƌƚŚĂŶĂĐƟŶŐĂƐĂsolo DNA-binding factor (29, 30, 50, 51). Towards ƉŽƚĞŶƟĂůŝŵƉůŝĐĂƟŽŶƐŽĨƚŚĞƐĞĮŶĚŝŶŐƐĨŽƌďƌĞĂƐƚĐĂŶĐĞƌďŝŽůŽŐLJ, GRHL2 has been ŝĚĞŶƟĮĞĚ ŝŶ ŵĂŝŶƚĂŝŶŝŶŐ ĞƉŝƚŚĞůŝĂů ŝĚĞŶƟƚLJ ĂŶĚ ŝŶ ŵŽĚƵůĂƟŶŐ Zɲ-ĚĞƉĞŶĚĞŶƚ ƚƌĂŶƐĐƌŝƉƟŽŶĂů programs, yet it has also been associated with worse clinical outcomes in ER-ƉŽƐŝƟǀĞƚƵŵŽƌƐ͘dŚĞ .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 25 ĮŶĞ-scale binding ƐƉĞĐŝĮĐŝƚLJ ƉĂƩĞƌŶƐ described here suggest that GRHL2 occupancy may be ŚŝŐŚůLJ ƐĞŶƐŝƟǀĞ ƚŽ ƐĞƋƵĞŶĐĞ ƉĞƌƚƵƌďĂƟŽŶƐ ǁŝƚŚŝŶ ƚŚĞcentral C dž dž ' Ž ƌ ƚ Ž Đ Ž Ž Ɖ Ğ ƌ Ă Ɵ ǀ Ğ Ɛ Ɖ Ă Đ ŝ Ŷ Ő  ƌĞůĂƟŽŶƐŚŝƉƐĂƚĚŝŵĞƌŝĐsites͘^ŽŵĂƟĐŵƵƚĂƟŽŶƐĂīĞĐƟŶŐƚŚĞƐĞƉŽƐŝƟŽŶƐ or CpG instability at GRHL2-enriched regions ĐŽƵůĚ ƐŚŝŌ 'Z,>Ϯ ďŝŶĚŝŶŐ ƉĂƩĞƌŶƐ ŝŶ ƚƵŵŽƌƐ͕ ƉŽƚĞŶƟĂůůLJ ǁĞĂŬĞŶŝŶŐ epithelial maintenance programs or altering ER-ĂƐƐŽĐŝĂƚĞĚĞŶŚĂŶĐĞƌĂĐƟǀŝƚLJ͘ŽŶǀĞƌƐĞůLJ͕ƌĞŐŝŽŶƐ ƚŚĂƚƌĞƚĂŝŶƐƚƌŽŶŐ'Z,>ϮďŝŶĚŝŶŐĐĂƉĂĐŝƚLJŵĂLJƌĞŝŶĨŽƌĐĞĞƉŝƚŚĞůŝĂůƚƌĂŶƐĐƌŝƉƟŽŶĂůƐƚĂƚĞƐĂnd could ƌĞƉƌĞƐĞŶƚ ĚŝƐƟŶĐƚ ƚŚĞƌĂƉĞƵƟĐ ƌĞƐƉŽŶƐĞ ĐŽŶƚĞdžƚƐ͘ ůƚŚŽƵŐŚ ŽƵƌ ƐƚƵĚLJ ĚŽĞƐ ŶŽƚ ĚŝƌĞĐƚůLJ ĂƐƐĞƐƐ ƚŚĞƐĞ ƌĞůĂƟŽŶƐŚŝƉƐ͕ ƚŚĞ ďŝŶĚŝŶŐparameters established here provide ŝŶĨŽƌŵĂƟŽŶ towards ŵĂƉƉŝŶŐŚŽǁƉĂƟĞŶƚ-ƐƉĞĐŝĮĐƐĞƋƵĞŶĐĞǀĂƌŝĂƟŽŶŵĂLJƌĞŵŽĚĞůƚŚĞ'Z,>ϮĐŝƐƚƌŽŵĞ͘ By analyzing GRHL2 binding at ChIP-seq sites in the absence of ĐŚƌŽŵĂƟŶĂŶĚĐŽŽƉĞƌĂƟǀĞ ƚƌĂŶƐĐƌŝƉƟŽŶĂů ĐŽ-regulators, this work provides a compendium of GRHL2 binding data for 770,000-plus genomic sites and ĚĞĮŶĞƐ, at an unprecedented high ƌĞƐŽůƵƟŽŶ, the sequence ƐƉĞĐŝĮĐŝƚLJ ƵŶĚĞƌůLJŝŶŐ 'Z,>Ϯ E ƌĞĐŽŐŶŝƟŽŶ ĂĐƌŽƐƐ ŐĞŶŽŵŝĐ ƐĞƋƵĞŶĐĞ ƐƉĂĐĞ͘The ability to resolve binding at sub-ƉĞĂŬ ƌĞƐŽůƵƟŽŶ͕ ƋƵĂŶƟĨLJ ƚŚĞ ĞīĞĐƚƐ ŽĨ ƐĞƋƵĞŶĐĞ ǀĂƌŝĂƟŽŶ͕ ĂŶĚ ĚĞĮŶĞ spacing-ĚĞƉĞŶĚĞŶƚŝŶƚĞƌĂĐƟŽŶƐĂĚǀĂŶĐĞƐŽƵƌƵŶĚĞƌƐƚĂŶĚŝŶŐ ŽĨ'Z,>ϮďĞLJŽŶĚĐŽŶƐĞŶƐƵƐŵŽƟĨ ŵŽĚĞůƐƚŽǁĂƌĚĂƉƌĞĚŝĐƟǀĞ͕ŵĞĐŚĂŶŝƐƟĐĚĞƐĐƌŝƉƟŽŶŽĨŝƚƐE-ďŝŶĚŝŶŐƐƉĞĐŝĮĐŝƚLJ͘ KƵƌĮŶĚŝŶŐƐ show that GRHL2 binding is anchored by a highly constrained central C dždžG within its octamer ĐŽŶƐĞŶƐƵƐŵŽƟĨand is ĂīĞĐƚĞĚ ďLJŶĞĂƌďLJŇĂŶŬŝŶŐƐĞƋƵĞŶĐĞ outside of its consensus. Our results support that GRHL2 ďŝŶĚŝŶŐŝƐƐƚƌĞŶŐƚŚĞŶĞĚĂƚĚŝŵĞƌŝĐƐŝƚĞƐĂŶĚĐĂŶďĞĚŝƐƟŶŐƵŝƐŚĞĚĂƐĞŝƚŚĞƌ direct DNA binding or cofactor-mediated occupancy across the genome. dŚĞƐĞŝŶƐŝŐŚƚƐƌĞĮŶĞthe structural and biochemical understanding of GRHL2 ŝŶƚĞƌĂĐƟŽŶǁŝƚŚE and lay the groundwork ĨŽƌ ĨƵƚƵƌĞ ƐƚƵĚŝĞƐ ůŝŶŬŝŶŐ Ɖ' ŵƵƚĂƟŽŶĂů ůĂŶĚƐĐĂƉĞƐ with GRHL2- ĚĞƉĞŶĚĞŶƚ ƚƌĂŶƐĐƌŝƉƟŽŶ ŝŶ .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 26 development and disease, as well as methods to disrupt key GRHL2 binding sites using site- ĚŝƌĞĐƚĞĚƚŚĞƌĂƉĞƵƟĐƐ.  .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 27 &/'hZ>'E^ &ŝŐƵƌĞϭ. &ƵŶĐƟŽŶĂůĚŽŵĂŝŶŽƌŐĂŶŝnjĂƟŽŶŽĨŚƵŵĂŶ'Z,>Ϯ͘ Domain structure of GRHL2 (isoform ϭͿ;ĐĐĞƐƐŝŽŶŶƵŵďĞƌEWͺϬϳϵϭϵϭ͘ϮͿ ƐŚŽǁŝŶŐƚŚĞƚƌĂŶƐĂĐƟǀĂƟŽŶĚŽŵĂŝŶ͕E-binding domain ;Ϳ͕ĐŽŶƐĞƌǀĞĚƐĞŐŵĞŶƚ͕ĂŶĚĚŝŵĞƌŝnjĂƟŽŶĚŽŵĂŝŶ, as adapted from (10). &ŝŐƵƌĞ Ϯ. Ğ Ɛ ŝ Ő Ŷ Ž Ĩ ƚ Ś Ğ ^ E  W Ă ƌ ƌ Ă LJ Ğ Ŷ Ă ď ů Ğ Ɛ Ś ŝ Ő Ś-ƌĞƐŽůƵƟŽŶ ŵĞĂƐƵƌĞŵĞŶƚ ŽĨ 'Z,>Ϯ ďŝŶĚŝŶŐ ĂĐƌŽƐƐ ŐĞŶŽŵŝĐ Ś/W-ƐĞƋ ƌĞŐŝŽŶƐ. Ϳ >ĞŌ͗ WƵƌŝĮĞĚ 'Z,>Ϯ ƉƌŽƚĞŝŶ ĂŶĚ ŇƵŽƌĞƐĐĞŶƚůLJ-labeled 'Z,>ϮĂŶƟďŽĚLJĂƐƐĂ LJ ĞĚŽŶƚŚĞ^EWĂƌƌ Ă LJ ͘DŝĚĚůĞ͗^ƵďƐĞĐƟŽŶŽĨƚŚĞ'Z,>ϮĂƌƌĂLJĚŝƐƉůĂLJŝŶŐ 'Z,>ϮǁŝƚŚĂŶƟďŽĚLJďŽƵŶĚƚŽEƉŽůLJŵĞƌĂƐĞĞdžƚĞŶĚĞĚ double-stranded probes. Right: Probes consisted of variable 48-nt genomic regions (green) with a 12-nt constant region (black) on the 3’ end ƵƐĞĚĨŽƌƉƌŝŵĞƌĞdžƚĞŶƐŝŽŶ͘dŚĞmicroarray strand ŝƐĂƩĂĐŚĞĚƚŽƚŚĞ^EWĂƌƌĂLJƐƵƌĨĂĐĞĂƚƚŚĞ 3' terminus. ͿPeak overlap of the four GRHL2 ChIP-seq datasets used to construct the genomic ƟůŝŶŐƌĞŐŝŽŶƐŽĨƚŚĞ^EWĂƌƌĂLJ͘ ͿClose-up of a region of the full SNAP array to show varying ŝŶƚĞŶƐŝƟĞƐ ŽĨ ŝŶĚŝǀŝĚƵĂů ďŽƵŶĚ ĨĞĂƚƵƌĞƐ. GRHL2 binding is detected as red circles. DͿ Top: GRHL2 ChIP-seq peak from Reese et al. (30) with the ƉŽƐŝƟŽŶ ŽĨ the OVOL1 gene displayed.  Ž Ʃ Ž ŵ ͗ ' Ğ Ŷ Ž ŵ ŝ Đ ^ E  W Ɖ ƌ Ž ď Ğ Ɛ Ɵ ů ŝ Ŷ Ő ƚ Ś Ğ ChIP-seq peak at 6-bp spacing with the ŶŽƌŵĂůŝnjĞĚ ^EW ŝŶƚĞŶƐŝƚLJ ĨŽƌ ĞĂĐŚ ƉƌŽďĞ ĚŝƐƉůĂLJĞĚ ƚŽ ƚŚĞ ƌŝŐŚƚ͘ dŚĞ ĐLJĂŶ ďŽdž ĚĞŶŽƚĞƐ Ă consensus GRHL2 site on the SNAP probe. The green bases in the SNAP probes correspond to genomic sequence while the black bases correspond to the constant primer. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 28 &ŝŐƵƌĞϯ͘^EWŐĞŶŽŵŝĐďŝŶĚŝŶŐĚĂƚĂƌĞĐŽǀĞƌƐƚŚĞ'Z,>ϮĐŽŶƐĞŶƐƵƐŵŽƟĨĂŶĚƌĞǀĞĂůƐŇĂŶŬŝŶŐ ƐĞƋƵĞŶĐĞ ƉƌĞĨĞƌĞŶĐĞƐ ĂŶĚ ŵŝƐŵĂƚĐŚ ĚŝƐƚƌŝďƵƟŽŶs. Ϳ džƉĞĐƚĂƟŽŶ ŵĂdžŝŵŝnjĂƟŽŶ ĂůŐŽƌŝƚŚŵ ŵŽƟĨƐĨŽƌƚŚĞŚŝŐŚĞƐƚŝŶƚĞŶƐŝƚLJ^EWŐĞŶŽŵŝĐƐĞƋƵĞŶĐĞƐǁŝƚŚĂĚĞĮŶĞĚŵŽƟĨǁŝĚƚŚŽĨϭϬďĂƐĞ pairs (bp). DŽƟĨĨƌŽŵƚŚĞƚŽƉϯϬϬ;ƚŽƉůĞŌͿ͕ϯϬϭ-600 (top right), 601-ϵϬϬ;ďŽƩŽŵůĞŌͿ͕ĂŶĚϵϬϭ- ϭϮϬϬ;ďŽƩŽŵright) highest intensity SNAP genomic sequences. WŽƐŝƟŽŶŶƵŵďĞƌŝŶŐŝƐďĂƐĞĚŽŶ the start of the GRHL2 octamer consensus, 5’-AACCGGTT-3’. Ϳ DŽƟĨŽĨƚŚĞϯϬϬŚŝŐŚĞƐƚŝŶƚĞŶƐŝƚLJ SNAP genomic sequences containing ĞdžĂĐƚůLJŽŶĞŵŝƐŵĂƚĐŚŝŶƚŚĞ'Z,>ϮĐŽŶƐĞŶƐƵƐŵŽƟĨ͘&ŝǀĞ ŇĂŶŬŝŶŐďĂƐĞƐĂƌĞŝŶĐůƵĚĞĚŝŶƚŚĞĚŝƐƉůĂLJĞĚŵŽƟĨ͘ͿŝƐĐƌŝŵŝŶĂƚŽƌLJĂŶĂůLJƐŝƐŽĨŇĂŶŬŝŶŐƐĞƋƵĞŶĐĞ surrounding the GRHL2 consensus using the highest versus lowest intensity genomic sequences. dŚŝƐ ƐĞƋƵĞŶĐĞ ƐĞƚ ŝƐ ĐŽŵƉŽƐĞĚ ĞdžĐůƵƐŝǀĞůLJ ŽĨ ŐĞŶŽŵŝĐ ƉƌŽďĞƐ ƚŚĂƚ ĐŽŶƚĂŝŶ ĂŶ ĞdžĂĐƚ ĐĂŶŽŶŝĐĂů GRHL2 consensus octamer, unlike ;-Ϳ. Top: A summary sequence of the consensus (black) and ĂŶĂůLJnjĞĚ ŇĂŶŬŝŶŐ ƐĞƋƵĞŶĐĞ ;ŐƌĞĞŶͿ ŝƐ ƉƌŽǀŝĚĞĚ͕ ǁŚĞƌĞ ƵŶĚĞƌƐĐŽƌĞƐ ŝŶĚŝĐĂƚĞ ŶŽ ƐŝŐŶŝĮĐĂnt ĚŝƐĐƌŝŵŝŶĂƚŽƌLJƉƌĞĨĞƌĞŶĐĞ͘ŽƩŽŵ͗dĂďůĞůŝƐƟŶŐĞŶƌŝĐŚĞĚďĂƐĞƐĂƚƐƉĞĐŝĮĐŇĂŶŬŝŶŐƉŽƐŝƟŽŶƐ͘dŚĞ ŶƵŵďĞƌŽĨĂƐƚĞƌŝƐŬƐĚĞŶŽƚĞƚŚĞƐŝŐŶŝĮĐĂŶĐĞŽĨƚŚĞƉ-value. Ϳ SSL of all SNAP genomic sequences ƵƐŝŶŐƚŚĞĐŽŶƐĞŶƐƵƐ'Z,>ϮŵŽƟĨ;ϱΖ-AACCGGTT-ϯΖͿĂƐƚŚĞĐĞŶƚƌĂů^^>ŵŽƟĨ͘Ϳ Two-dimensional ƌĞƉƌĞƐĞŶƚĂƟŽŶ ŽĨ ƚŚĞ ^^> ϭ-mismatch ring. This ring is divided into regions corresponding to where the mismatch to the GRHL2 consensus occurs. Genomically, mismatches in the central two ƉŽƐŝƟŽŶƐŽĨƚŚĞŵŽƟĨŽĐĐƵƌŵƵch more commonly (~10- to 20-ĨŽůĚͿƚŚĂŶƚŚĞŽƚŚĞƌƉŽƐŝƟŽŶƐ͘dŚĞ Ŷ Ƶ ŵ ď Ğ ƌ Ž Ĩ Ɖ ƌ Ž ď Ğ Ɛ Ă ƚ ƚ Ś Ğ Ě ŝ ī Ğ ƌ ŝ Ŷ Ő ŵ ŝ Ɛ ŵ Ă ƚ Đ Ś Ɖ Ž Ɛ ŝ Ɵ Ž Ŷ Ɛ ŝ Ɛ Ɛ LJ ŵ ŵ Ğ ƚ ƌ ŝ ĐĂ ů Ɛ ŝ Ŷ Đ Ğ ƚ Ś Ğ ŵ Ž Ɵ Ĩ ŝ Ɛ  palindromic. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 29 &ŝŐƵƌĞϰ.^LJƐƚĞŵĂƟĐŵŝƐŵĂƚĐŚĂŶĂůLJƐŝƐƌĞǀĞĂůƐƐĞƋƵĞŶĐĞƌƵůĞƐŐŽǀĞƌŶŝŶŐ'Z,>ϮŵŽŶŽŵĞƌĂŶĚ ĚŝŵĞƌďŝŶĚŝŶŐ. Ϳ džƉĞĐƚĂƟŽŶŵĂdžŝŵŝnjĂƟŽŶĂůŐŽƌŝƚŚŵŵŽƟĨĨŽƌƚŚĞϭϮϬϬŚŝŐŚĞƐƚŝŶƚĞŶƐŝƚLJ^EW ŐĞŶŽŵŝĐƐĞƋƵĞŶĐĞƐǁŝƚŚĂĚĞĮŶĞĚŵŽƟĨǁŝĚƚŚŽĨϭϴďĂƐĞƉĂŝƌƐ;ďƉͿ͘ͿCrystal structure of GRHL2 DNA-binding domain (PDB͗ϱDZϳͿǁŝƚŚŽǀĞƌůĂŝĚůŝŶĞĂƌĚƵƉůĞdžEƐĐŚĞŵĂƟĐƉůĂĐĞĚŽŶƚŚĞďĂƐŝƐ of the GRHL1-DNA crystal structure (PDB: 5MPF). ͿAverage normalized SNAP intensity for an increasing number of mismatches to the consensus sequence 5’-AACCGGTT-3’. Each mismatch group represents all possible mismatches to the consensus͕ƌĞƐƵůƟŶŐŝŶϮϰƵŶŝƋƵĞŽŶĞ-mismatch probes, 252 unique two-mismatch probes, 1512 unique three-mismatch probes, and 5670 unique four-mismatch probes, placed on the SNAP array in triplicate͘dŚĞŵŽƟĨƐĂƌĞĂŶĐŚŽƌĞĚŽŶĞŝƚŚĞƌ side by constant regions (C1 and C2). Error bars ;ƌĞƉƌĞƐĞŶƟŶŐŽŶĞƐƚĂŶĚĂƌĚĚĞǀŝĂƟŽŶͿare derived ĨƌŽŵ Ăůů ŝŶƚĞŶƐŝƟĞƐ ŽĨ Ăůů ƌĞƉůŝĐĂƚĞ ƉƌŽďĞƐ ĂŶĚ ƉŽƐƐŝďůĞ ŵŝƐŵĂƚĐŚ ǀĂƌŝĂƟŽŶƐ ǁŝƚŚŝŶ ƚŚĞ group. Ϳ īĞĐƚƐŽĨŵŝƐŵĂƚĐŚ(es) in a dimeric GRHL2 consensus site separated by a single base ƉĂŝƌ͕ŇĂŶŬĞĚďLJĐŽŶƐƚĂŶƚƌĞŐŝŽŶƐ͘dŚĞdž-ĂdžŝƐůĂďĞůƐĚĞŶŽƚĞƚŚĞŶƵŵďĞƌŽĨŵŝƐŵĂƚĐŚĞƐŝŶƚŚĞůĞŌ binding site followed by the number of mismatch(e s) in the right binding site, separated by an unĚĞƌƐĐŽƌĞ͘ĂĐŚƉƌŽďĞƌĞƉƌĞƐĞŶƚĞĚŝŶƚŚĞĮƌƐƚĮǀĞ ĐŽůƵŵŶƐĐŽŶƐŝƐƚƐŽĨŝĚĞŶƟĐĂů mismatches in the two binding sites, while the middle and last four columns consist of mismatch(es) only in the ĮƌƐƚ Žƌ ƐĞĐŽŶĚ ďŝŶĚŝŶŐ ƐŝƚĞ͕ ƌĞƐƉĞĐƟǀĞůLJ͕ ǁŝƚŚ ƚŚĞ ŽƚŚĞƌ ďŝŶĚŝŶŐ Ɛ ŝ ƚ Ğ Đ Ž ŵ Ɖ ƌ ŝ Ɛ Ğ Ě Ž Ĩ ƚ Ś Ğ  consensus. Ϳ ǀĞƌĂŐĞ ŶŽƌŵĂůŝnjĞĚ ^EW ŝŶƚĞŶƐŝƟĞƐ ĨŽƌ ĞĂĐŚ ƐŝŶŐůĞ ŵŝƐŵĂƚĐŚ ƚŽƚŚĞ 'Z,>Ϯ ĐŽŶƐĞŶƐƵƐƐŝƚĞďĂƐĞĚŽŶĐŽŶƚƌŽůƉƌŽďĞŝŶƚĞŶƐŝƟĞƐ͘ Red bases denote mismatches to the GRHL2 consensus. &Ϳ ǀĞƌĂŐĞŶŽƌŵĂůŝnjĞĚ^EWŝŶƚĞŶƐŝƟĞƐĨŽƌƚŚĞƐŝdž highest and seven lowest intensity ŵŝƐŵĂƚĐŚŵŽƟĨƐǁŚĞƌĞƚŚĞŶƵŵďĞƌŽĨŵŝƐŵĂƚĐŚĞƐ to the canonical GR,>ϮŵŽƟĨ vary up to four. Red bases denote mismatches to the GRHL2 consensus. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 30 &ŝŐƵƌĞ ϱ͘ 'Z,>Ϯ ĚŝŵĞƌ ďŝŶĚŝŶŐ ƐƚƌĞŶŐƚŚ ŽƐĐŝůůĂƚĞƐ ǁŝƚŚ E ŚĞůŝĐĂů ƉĞƌŝŽĚŝĐŝƚLJ͘ Ϳ Mean ŇƵŽƌĞƐĐĞŶĐĞŝŶƚĞŶƐŝƚLJ;ц^DͿĨŽƌĨĞĂƚƵƌĞƐĐŽŶƚĂŝŶŝŶŐƚǁŽ'Z,>ϮŵŽƟĨƐƐĞƉĂƌĂƚĞĚďLJϬ-32 bp in forward-forward (gray) or reverse-forward (whiteͿŽƌŝĞŶƚĂƟŽŶ͕ŝůůƵƐƚƌĂƟŶŐŽƌŝĞŶƚĂƟŽŶ-dependent ĚŝīĞƌĞŶĐĞƐ ŝŶ ďŝŶĚŝŶŐ ƐŝŐŶĂů͘Ϳ Forward-ĨŽƌǁĂƌĚ ŇƵŽƌĞƐĐĞŶĐĞ ǀĂůƵĞƐ ƉůŽƩĞĚ ŽŶ Ă ůŽŐĂƌŝƚŚŵŝĐ Ɛ Đ ĂůĞĂƐĂĨ ƵŶĐƟ Ž ŶŽ ĨĐĞŶ ƚ Ğƌ-to-ĐĞŶƚĞƌŵŽƟĨƐƉĂĐŝŶŐ͘WŽŝŶƚƐƌĞƉƌĞƐĞŶƚ ŵĞĂŶц^D͘dŚĞďůĂĐŬ curve shows the best-ĮƚƚŽƌƐŝŽŶĂůƉŚĂƐŝŶŐŵŽĚĞů͕LJŝĞůĚŝŶŐƉĞƌŝŽĚŝĐŝƚLJŽĨĂƉƉƌŽdžŝŵĂƚĞůLJϱ͘ϰϲďƉ͘  Ă ƚ Ă Ɖ Ž ŝ Ŷ ƚ Ɛ Ă ƌ Ğ Đ Ž ů Ž ƌ Ğ Ě ď LJ ƚ Ś Ğ Ɖ Ś Ă Ɛ Ğ Ž Ĩ ƚ Ś Ğ Į Ʃ Ğ Ě Ś Ğ ů ŝ Đ Ă ů Ɖ Ğ ƌ ŝ Ž ĚŝĐŝƚLJ ŵŽĚĞů͘Ϳ Helical ƌĞƉƌĞƐĞŶƚĂƟŽŶŽĨƚŚĞĨŽƌǁĂƌĚ-ĨŽƌǁĂƌĚĚĂƚĂ͕ŵĂƉƉŝŶŐŵŽƟĨƐƉĂĐŝŶŐŽŶƚŽƚŚĞĞdžƉĞĐƚĞĚŐĞŽŵĞƚƌLJ ŽĨƚŚĞEŚĞůŝdž;ϭϬ͘ϱďƉƉĞƌƚƵƌŶͿ͘WŽŝŶƚƐƌĞƚĂŝŶĐŽůŽƌĐŽĚŝŶŐĨƌŽŵƉĂŶĞů(Ϳ͕ŚŝŐŚůŝŐŚƟŶŐƚŚĂƚ ƉĞĂŬďŝŶĚŝŶŐŝŶƚĞŶƐŝƟĞƐĐůƵƐƚĞƌŽŶĂůƚĞƌŶĂƟŶŐĨĂĐĞƐŽĨƚŚĞEŚĞůŝdž͕ĐŽŶƐŝƐƚĞŶƚǁŝƚŚƉƌĞĨĞƌĞŶƟĂů binding when GRHL2 dimers occupy the same helical face. Ϳ Structural model ŝůůƵƐƚƌĂƟŶŐƚŚĞ ƉƌĞĚŝĐƚĞĚ ĐŽŶĮŐƵƌĂƟŽŶ Ăƚ ƚŚĞ ŽƉƟŵĂů ĨŽƌǁĂƌĚ-forward spacing of 1 bp. Using the crystal structure of GRHL1 dimer bound to DNA, two GRHL2 dimers, depicted as yellow and salmon monomers forming dimer 1 and white and blue monomers forming dimer 2, ǁĞƌĞƉŽƐŝƟŽŶĞĚƚŽ ŵŽĚĞůďŝŶĚŝŶŐƚŽƚǁŽEŵŽƟĨƐƐƉĂĐĞĚϭďƉĂƉĂƌƚ͘  &ŝŐƵƌĞϲ͘^EWĚĂƚĂĚŝƐƟŶŐƵŝƐŚĞĚĚŝƌĞĐƚ'Z,>ϮEďŝŶĚŝŶŐĨƌŽŵŝŶĚŝƌĞĐƚƉƌŽƚĞŝŶ-ŵĞĚŝĂƚĞĚ ƌĞĐƌƵŝƚŵĞŶƚ Ăƚ ŐĞŶŽŵŝĐ ƐŝƚĞƐ. Ϳ ^ĐŚĞŵĂƟĐ ĚĞŵŽŶƐƚƌĂƟŶŐ ƚŚĞ ŵĞƚŚŽĚ ƚŽ ĚĞƚĞƌŵŝŶĞ ĚŝƌĞĐƚ ǀĞƌƐƵƐŝŶĚŝƌĞĐƚďŝŶĚŝŶŐŽĨ'Z,>ϮĂƚŐĞŶŽŵŝĐƐŝƚĞƐ͘>ĞŌ͗tŚĞŶďŝŶĚŝŶŐŝƐŽďƐĞƌǀĞĚďLJďŽƚŚŚ/W- ƐĞƋĂŶĚ^EWĂƌƌĂLJƐĂƚƚŚĞƐĂŵĞŐĞŶŽŵŝĐůŽĐĂƟŽŶ͕ƚŚŝƐŝƐĐŽŶƐŝĚĞƌĞĚĂĚŝƌĞĐƚďŝŶĚŝŶŐĞǀĞŶƚ͘ZŝŐŚƚ͗ When binding is observed by ChIP-seq but not SNAP arrays, then the ChIP-seq binding is considered to be mediated by a secondary cofactor (CoF) that indirectly tethers GRHL2 to this .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 31 ŐĞŶŽŵŝĐůŽĐĂƟŽŶ͘ͿZĞƉƌĞƐĞŶƚĂƟǀĞĞdžĂŵƉůĞƐŽĨĂĚŝƌĞĐƚ;>ĞŌͿĂŶĚŝŶĚŝƌĞĐƚ;ZŝŐŚƚͿďŝŶĚŝŶŐĞǀĞŶƚ͘ ChIP-ƐĞƋƐŝŐŶĂůĨƌŽŵŽǀĞƌĞdžƉƌĞƐƐĞĚ;dŽƉͿĂŶĚĞŶĚŽŐĞŶŽƵƐ;DŝĚĚůĞͿ'Z,>ϮůĞǀĞůƐĂƚƚŚĞDd^ϭ and EPIC1 promoters (7, 30) ͘ EŽƌŵĂůŝnjĞĚ 'Z,>Ϯ ďŝŶĚŝŶŐ ŝŶƚĞŶƐŝƚLJ ;ŽƩŽŵͿis shown at these promoters ĂƐƟůĞĚŽŶƚŚĞ^EWĂƌƌĂLJ͘ĂĐŚƌĞŐŝŽŶĐŽŶƚĂŝŶƐŵƵůƟƉůĞEƉƌŽďĞƐƟůĞĚĂĐƌŽƐƐƚŚĞ region. ͿSummary of the genomic probe count on the SNAP array with the corresponding number of genomic regions evaluated using overlapping SNAP probes. These genomic regions are then divided into direct and indirect GRHL2 site s based on the SNAP probe in each region with the highest intensity. Ϳ Ăƌ ŐƌĂƉŚ ŽĨ ƚŚĞ ŶƵŵďĞƌ ŽĨ ĚŝƐƟŶĐƚ 'Z,>Ϯ ďŝŶĚŝŶŐ Ɛites within each ŐĞŶŽŵŝĐƌĞŐŝŽŶƟůĞĚďLJƚŚĞ^EWĂƌƌĂLJ͘KŶĞƌĞŐŝŽŶ͕ĐŚƌϳ͗ϭϱϱ͕ϭϮϯ͕ϰϬϬ-155,125,400 (Figure 7), ǁĂƐĞdžĐůƵĚĞĚĨƌŽŵƚŚŝƐĂŶĂůLJƐŝƐĚƵĞĂůĂƌŐĞŶƵŵďĞƌŽĨĚŝƌĞĐƚďŝŶĚŝŶŐƐŝƚĞƐǁŝƚŚŝŶĂǀĞƌLJƐŚŽƌƚ region, where numerous SNAP ƉƌŽďĞƐĐŽŶƚĂŝŶŵƵůƟƉůĞ'Z,>ϮĚŝƌĞĐƚďŝŶĚŝŶŐƐŝƚĞƐ͘ &ŝŐƵƌĞϳ͘ŐĞŶŽŵŝĐƌĞŐŝŽŶǁŝƚŚĞdžƚƌĞŵĞ'Z,>ϮŵŽƟĨĚĞŶƐŝƚLJƉƌŽĚƵĐĞƐďƌŽĂĚ^EWďŝŶĚŝŶŐ ƐŝŐŶĂůƚŚĂƚŝƐŶŽƚĨƵůůLJƌĞŇĞĐƚĞĚŝŶŚ/W-ƐĞƋƉƌŽĮůĞƐ. Ϳ Genomic sequence of chr7:155,124,004- ϭϱϱ͕ϭϮϰ͕ϳϱϵ ;'ZŚϯϴͿ͘ ŽŶƐĞŶƐƵƐ ŵŽƟĨƐ ĂƌĞ ƵŶĚĞƌůŝŶĞĚ ǁŝƚŚ ƉĞƌĨĞĐƚ ƐŝƚĞƐ ŝŶ ƌĞĚ ĂŶĚ ƐŝŶŐůĞ ŵŝƐŵĂƚĐŚƐŝƚĞƐŝŶďůƵĞ͘>ŽǁĐŽŵƉůĞdžŝƚLJƌĞƉĞĂƚƐŽĨϮϮďƉƐƚĂƌƚĂŶĚĞŶĚǁŝƚŚƚŚĞďĂƐĞƐŝŶLJĞůůŽǁ highlight. Ϳ Top: ChIP-seq data from Zheng et al. (7) and Reese et al. (30) are displayed for this ƌĞŐŝŽŶ͘ŽƩŽŵ͗EŽƌŵĂůŝnjĞĚ^EWŝŶƚĞŶƐŝƟĞƐĨŽƌŐĞŶŽŵŝĐƉƌŽďĞƐĂĐƌŽƐƐƚŚŝƐƌĞŐŝŽŶƟůĞĚĂƚevery 6 bp are displayed for this region.  .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 32 'DEd^ The authors thank members of the Fowler Laboratory, Alarid Laboratory, and Proteovista LLC for their helpful comments on the data and manuscript. /ŶƉĂƌƟĐƵůĂƌ͕ǁĞƚŚĂŶŬEĂƚĂƐŚĂ^ŽůŽĚŝŶ͕ Blue-leaf Cordes, Kelley Salem, Kaelyn Allen, and Christy Zheng ĨŽƌƐĐŝĞŶƟĮĐĚŝƐĐƵƐƐŝŽŶƐ͘  &hE/E' This research was ƐƵƉƉŽƌƚĞĚďLJEĂƟŽŶĂů/ŶƐƟƚƵƚĞƐof Health͕EĂƟŽŶĂůĂŶĐĞƌ/ŶƐƟƚƵƚĞ [grant number R01 CA260140 to ETA and AMF] with a subaward to Proteovista LLC. The funders had ŶŽƌŽůĞŝŶƚŚĞĚĞƐŝŐŶŽĨƚŚŝƐƐƚƵĚLJ͖ŝŶƚŚĞĐŽůůĞĐƟŽŶ͕ĂŶĂůLJƐĞƐ͕ŽƌŝŶƚĞƌƉƌĞƚĂƟŽŶŽĨĚĂƚĂ͖ŝŶƚŚĞ ǁƌŝƟŶŐŽĨƚŚĞŵĂŶƵƐĐƌŝƉƚ͖ŽƌŝŶƚŚĞĚĞĐŝƐŝŽŶƚŽƉƵďůŝƐŚƚŚĞƌĞƐƵůƚƐ͘ Funding for open access ĐŚĂƌŐĞ͗EĂƟŽŶĂů/ŶƐƟƚƵƚĞƐŽĨ,ĞĂůƚŚ͘ KE&>/dK&/EdZ^d These authors have the following roles at Proteovista LLC: Mary S. Ozers is Co-Founder, Owner, ĂŶĚŚŝĞĨ^ĐŝĞŶƟĮĐKĸĐĞƌ; Christopher L. Warren is Co-Founder and Chief Biotechnology KĸĐĞƌ͖WĂŝŐĞE. Messa, employee; Noah R. Nicol, employee; Keenan S. Pearson, employee. Amy M. &ŽǁůĞƌƌĞĐĞŝǀĞƐŬĐŚĂƉƚĞƌƌŽLJĂůƚLJĨƌŽŵůƐĞǀŝĞƌ͕/ŶĐ͘KƚŚĞƌĂƵƚŚŽƌƐĚĞĐůĂƌĞŶŽĐŽŶŇŝĐƚŽĨ interest. hd,KZKEdZ/hd/KE^ ŽŶĐĞƉƚƵĂůŝnjĂƟŽŶ͗DĂƌLJ^͘KnjĞƌƐ͕ůĂŝŶĞd ͘ůĂƌŝĚ, Christopher L. Warren, Paige E. Messa, Keenan S. Pearson ĂƚĂƵƌĂƟŽŶ͗WĂŝŐĞE. Messa, Christopher L. Warren, Noah R. Nicol Formal Analysis: Christopher L. Warren, Paige E. Messa, Noah R. Nicol, Keenan S. Pearson͕:ƵƐƟŶ P. Pe t e r s &ƵŶĚŝŶŐĂĐƋƵŝƐŝƟŽŶ͗ůĂŝŶĞd ͘ůĂƌŝĚ͕ŵLJD͘&ŽǁůĞƌ͕DĂƌLJ^͘KnjĞƌƐ /ŶǀĞƐƟŐĂƟŽŶ͗ Paige E. Messa, Keenan S. Pearson, Christopher L. Warren, Noah R. Nicol Methodology: Paige E. Messa, Keenan S. Pearson, Christopher L. Warren, Noah R. Nicol, Mary S. Ozers WƌŽũĞĐƚĚŵŝŶŝƐƚƌĂƟŽŶ͗ Mary S. Ozers, Elaine T. Alarid Resources: Mary S. Ozers ^ŽŌǁĂƌĞ͗ Noah R. Nicol, Christopher L. Warren Supervision: Mary S. Ozers, Elaine T. Alarid, Amy M. Fowler sĂůŝĚĂƟŽŶ͗ Paige E. Messa, Keenan S. Pearson, Christopher L. Warren, Noah R. Nicol sŝƐƵĂůŝnjĂƟŽŶ͗ Paige E. Messa, Keenan S. Pearson, Christopher L. Warren, Noah R. Nicol, Mary S. Ozers tƌŝƟŶŐ– ŽƌŝŐŝŶĂůĚƌĂŌ: Paige E. Messa, Mary S. Ozers tƌŝƟŶŐ– ƌĞǀŝĞǁΘĞĚŝƟŶŐ͗WĂŝŐĞE. Messa, Christopher L. Warren, Noah R. Nicol, Keenan S. WĞĂƌƐŽŶ͕:ƵƐƟŶW ͘WĞƚĞƌƐ͕ŵLJD͘&ŽǁůĞƌ͕ůĂŝŶĞd ͘ůĂƌŝĚ͕DĂƌLJ^͘KnjĞƌƐ  .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Comprehensive Genomic Analysis Reveals that the WŝŽŶĞĞƌŝŶŐ&ƵŶĐƟŽŶŽĨ&Kyϭ/Ɛ/ŶĚĞƉĞŶĚĞŶƚŽĨ,ŽƌŵŽŶĂů^ŝŐŶĂůŝŶŐ͘ĞůůZĞƉ͘ 2019;26(10):2558-ϲϱ͘Ğϯ͘ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐĞůƌĞƉ͘ϮϬϭϵ͘ϬϮ͘Ϭϯϲ͘WƵďDĞĚWD/͗ϯϬϴϰϬϴϴϭ͖WƵďDĞĚ Central PMCID: PMC6408623. 49. 'ůŽŶƚ^͕WĂƉĂĐŚƌŝƐƚŽƵ<͕^ĂǁůĞ͕,ŽůŵĞƐŽ^KŶĞ͘ ϮϬϭϵ͖ϭϰ;ϰͿ͗ĞϬϮϭϱϯϰϬ͘ƉƵďϮϬϭϵϬϰϭϬ͘ĚŽŝ͗ϭϬ͘ϭϯϳϭͬũŽƵƌŶĂů͘ƉŽŶĞ͘ϬϮϭϱϯϰϬ͘WƵďDĞĚWD/͗ 30970003; PubMed Central PMCID: PMC6457525. 50. ĂƌƚƐDd ͕EŽƌĚŝŶ͕ĂŶƚƶ͕ǀĂŶŽdžƚĞů>͕ǀĂŶŵĞƌŽŶŐĞŶZ͘DĞĐŚĂŶŝƐƟĐĚŝƐƐĞĐƟŽŶŽĨ 'Z,>ϮĂŶĚWZƚƌĂŶƐĐƌŝƉƟŽŶĂůĐŽ-ƌĞŐƵůĂƟŽŶŝŶďƌĞĂƐƚĐĞůůƐ͘W>Ž^'ĞŶĞƚ͘ϮϬϮϲ͖ϮϮ;ϯͿ͗ĞϭϬϭϮϬϴϴ͘ ƉƵďϮϬϮϲϬϯϭϳ͘ĚŽŝ͗ϭϬ͘ϭϯϳϭͬũŽƵƌŶĂů͘ƉŐĞŶ͘ϭϬϭϮϬϴϴ͘WƵďDĞĚWD/͗ϰϭϴϰϯϲϮϮ͖WƵbMed Central PMCID: PMC13008249. 51. Mohammed H, Taylor C, Brown GD, Papach ristou EK, Carroll JS, D'Santos CS. Rapid ŝŵŵƵŶŽƉƌĞĐŝƉŝƚĂƟŽŶŵĂƐƐƐƉĞĐƚƌŽŵĞƚƌLJŽĨĞŶĚŽŐĞŶŽƵƐƉƌŽƚĞŝŶƐ;Z/DͿĨŽƌĂŶĂůLJƐŝƐŽĨ ĐŚƌŽŵĂƟŶĐŽŵƉůĞdžĞƐ͘EĂƚWƌŽƚŽĐ͘ϮϬϭϲ͖ϭϭ;ϮͿ͗ϯϭϲ-Ϯϲ͘ƉƵďϮϬϭϲͬϬϭͬϮϭ͘ĚŽŝ͗ ϭϬ͘ϭϬϯϴͬŶƉƌŽƚ͘ϮϬϭ6.020. PubMed PMID: 26797456. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint Graphical Abstract .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint GRHL2 625 aa Transactivation domain DNA binding domain (DBD) Conserved DBD segment Dimerization domain 1 1 3 3 2 4 7 4 8 4 ~ 4 9 4 6 2 5 416 424 Figure 1 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint A B 9.163 11.826 3.003 0.879 SNAP Fluorescence Intensity SNAP Array Sequence Chromosome 11 ………………………………………………..... ChIP-Seq 0.532 4.577 OVOL1 5’-CCCACCTATTTGTTACCTGTCGAACCGGTTTCCATTCCGCTGCGGGTGCATCGCAAGCCT-3’ 3’-GGGTGGATAAACAATGGACAGCTTGGCCAAAGGTAAGGCGACGCCCACGTAGCGTTCGGA-5’ 5’-TGACAACCCACCTATTTGTTACCTGTCGAACCGGTTTCCATTCCGCTGCATCGCAAGCCT-3’ 3’-ACTGTTGGGTGGATAAACAATGGACAGCTTGGCCAAAGGTAAGGCGACGTAGCGTTCGGA-5’ 5’-TAACGGTGACAACCCACCTATTTGTTACCTGTCGAACCGGTTTCCATTCATCGCAAGCCT-3’ 3’-ATTGCCACTGTTGGGTGGATAAACAATGGACAGCTTGGCCAAAGGTAAGTAGCGTTCGGA-5’ 5’-GGGAAATAACGGTGACAACCCACCTATTTGTTACCTGTCGAACCGGTTCATCGCAAGCCT-3’ 3’-CCCTTTATTGCCACTGTTGGGTGGATAAACAATGGACAGCTTGGCCAAGTAGCGTTCGGA-5’ P3: chr11_65786865-65786912 P2: chr11_65786859-65786906 P4: chr11_65786871-65786918 P5: chr11_65786877-65786924 5’-TGCGTTGGGAAATAACGGTGACAACCCACCTATTTGTTACCTGTCGAACATCGCAAGCCT-3’ 3’-ACGCAACCCTTTATTGCCACTGTTGGGTGGATAAACAATGGACAGCTTGTAGCGTTCGGA-5’ P6: chr11_65786883-65786930 P1: chr11_65786853-65786900 Add purified GRHL2 to SNAP array Detect on SNAP array Genomic sequence Primer Linker Slide 5’-TATTTGTTACCTGTCGAACCGGTTTCCATTCCGCTGCGGGTGAGGTGGCATCGCAAGCCT-3’ 3’-ATAAACAATGGACAGCTTGGCCAAAGGTAAGGCGACGCCCACTCCACCGTAGCGTTCGGA-5’ D Array features each containing 105 –1 06 copies of dsDNA probes968,014 total DNA features C Bound features dsDNA probes Figure 2 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint 0 5 10 15 20 0 5 10 15 20 _GA AACCGGTT C_GAG 1 2 4 5 7 8 B C E Position 1 A 1,984 mutations (4.4%) Position 2 A 1,917 mutations (4.2%) Position 3 C 861 mutations (1.9%) Position 4 C 17,930 mutations (39.5%) Position 5 G 17,930 mutations (39.5%) Position 6 G 861 mutations (1.9%) Position 7 T 1,917 mutations (4.2%) Position 8 T 1,984 mutations (4.4%) Dip in 0 mm ring: Consensus at either end of 48mer (Edge or next to primer) CA repeats (low complexity) TA repeats followed by GA repeats (low complexity) AACNGGTT (middle area of probe) AACNNGTT (best = AACWWGTT) D A 1-300 301-600 Position Bits 0 1 2 601-900 901-1200 Position Bits 0 1 2 Position Bits 0 1 2 Position Bits 0 1 -1 1 2 3 4 2 5678 + 1 3 6__ Location Preferred Base Enrichment Ratio P-value 2 bp upstream 1 bp upstream 1 bp downstream 3 bp downstream 4 bp downstream 5 bp downstream G A C G A G 1.55x 1.71x 1.59x 1.28x 1.62x 1.44x <0.001*** <0.001*** 0.003** 0.042* <0.001*** <0.001*** Position Bits 0 1 -5 -4 -3 -2 -1 2 1 2 3 4 5 6 7 8 +1 +2 +3 +4 +5 Figure 3 - 1123456 78-2 - 112345678-2 - 112345678-2 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint C1-AACCGGTT-C2 Mismatches Normalized Intensity Consensus 1 2 3 4 2 4 6 0 BC Mismatches (Left_Right) Normalized Intensity 1_1 2_2 3_3 4_4 1_0 2_0 0_3 0_40_1 0_23_0 4_0 10 0 D C1-AACCGGTT-A-AACCGGTT-C2 Motif Normalized Intensity GACCGGTT 0 2 4 6 TACCGGTT AACGGGTT CACCGGTT AACCGTTT AACCGGAT AACCTGTT AGCCGGTT ACCCGGTT AACTGGTT AATCGGTT AACCGGTT E Normalized Intensity 0 2 4 6 AACCGGTT AACCGGTC AACCGGTA TACCGGTA GGCCGGCA GGCCGGCC GACCGGCC CCCCCGTA CAGCGGCC AAACTTTG ATGCCGTA AAGCAGAG ATCCCCTG ATGCCGTG F 1-1200 Position Bits 0 1 2345 2 67 8 12345678 A Figure 4 Motif 1 Motif 2 0_0 30 20 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint Figure 5 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint A chrX:120,603,600-120,604,450 Normalized Tag Density Endogenous GRHL2 GRHL2 Over- expression 200 bp MCTS1 25 150 0 10 20 30 SNAP Array Relative GRHL2 Binding Intensity Direct Binding Event Indirect Binding Event chr22:47,631,400-47,632,250 25 150 200 bp EPIC1 0 10 20 30 SNAP Array Relative GRHL2 Binding Intensity B 772,732 Genomic SNAP Probes 6,151 Genomic Regions 4,566 GRHL2 Bound Direct binding 1,585 Unbound Indirect binding C 4000 3000 2000 1000 0 5102 3 4 1,585 3,814 668 72 11 1 Number of Distinct GRHL2 Binding Events Number of Genomic Regions Number of Distinct GRHL2 Binding Events In SNAP Array Genomic Regions D Direct Binding Event Indirect Binding Event SNAP Array: Signal: YES Signal: NOSignal: YES ChIP-seq: SNAP Array: ChIP-seq: Signal: YES Normalized Tag Density Figure 6 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint A B ATTGCTTGGCTGAGGTTGCCAGGTTTCTCCACCGTAAAGTTACTTTTT TTTTTTTTAAAGGTGTGCAAATTAAATGAGTGGTTTCTCATAGTCAAAC AACCGGTTTCTCATAGCCAAACAACCGGTTTCTCATAGCCAAACAAC CGGTTTCTCATAGTCAAACAACCGGTTTCTCATAGTCAAACAACCGG TTTCTCATAGTCAAACAACCGGTTTCTCATAGCCAAACAACCGGTTTC TCATAGTCAAACAACCGGTTTCTCATAGTCAAACAACCGGTTTCTCAT AGTCAAACAACCGGTTTCTAATAGTCAAACAACCGGTTTCTCATAGTC AAACAACCGGTTTCTCATAGCCAAACAACCGGTTTCTCATAGCCAAA CAACCGGTTTCTCATAGTCAAACAACTGGTTTCTCATAGTCAAACAAC CGGTTTCTCATAGCCAAACAACCGGTTTCTCATAGCCAAACAACCGG TTTCTCATAGTCAAACAACCGGTTTCTCATAGTCAAACAACCGGTTTC TCATAGTCAAACAACCGGTTTCTCATAGTCAAACAAACGGTTTCTCAT AGTCAAACAACCGGTTTCTCATAGTCAAACAACCGGTTTCTCATAGTC AAACAAACGGTTTCTCATAGTCAAACAACCGGTTTCTAATAGTCAAAC ACTTTCCTCTGGGCTGGCTTCATTCTCATGCTCTCAGTGGCAAAGAG AGCTCCTAGTAGAAAACTCAATGAGAAAAGTGAAGAACA chr7:155,123,400-155,125,400 Normalized Tag Density Endogenous GRHL2 GRHL2 24-hour Induction 200 bp 25 150 10 20 30 SNAP Array Relative GRHL2 Binding Intensity Figure 7 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 18, 2026. ; https://doi.org/10.64898/2026.04.16.719077doi: bioRxiv preprint