Thread by @TheDoctorIsVin

Very excited to share my work on Stag2, chromatin architecture, and effects on hematopoiesis! Join me in a @MedTweetorials of why this is so important! @CellStemCell cell.com/cell-stem-cell…

The cohesin complex is comprised of structural proteins (Smc1a, Smc3, Rad21) bound to a Stag protein. We and others have described that alterations of each affect hematopoietic self-renewal and differentiation.

However, both the mechanism and the biologic reason for the overwhelming prevalence of STAG2 being overwhelmingly the most commonly mutated component in cancer was still not known.

How does the cohesin complex affect 3-dimensional DNA structure and how does this affect transcriptional regulation? What is the difference between loss of a structural cohesin versus a Stag protein?

Using novel conditional KO mice (now available @jacksonlab), we show major hematologic defects (myeloid skew, B cell loss) with Stag2 loss, but not in Stag1. Co-deletion is lethal (shown by @johannes_zuber and colleagues); Stag1 redundancy must maintain viability, but how?

ChIPseq for Stag2 and Stag1 in HSPC of WT and Stag2 KO mice shows both common and Stag2-unique binding sites with Stag1 able to bind TAD boundaries. If Stag1 rescue maintains TAD integrity, is structural integrity lost where Stag1 fails to bind?

Genes with decreased expression and decreased ATAC accessibility lose short range DNA-DNA interactions by HiC (10kB resolution), including key B cell lineage commitment genes, including PU.1 programs. Is this a loss of PU.1 expression or a loss of activity?

PU.1 expression is preserved in MPP and CLP populations, but ChIPseq for PU.1 shows decreased PU.1 binding genome wide. But does failed expression impair DNA structure or does DNA structural changes impair transcriptional programming?

The PU.1-target, Ebf1, loses insulation, accessibility, and expression. As PU.1 is a putative pioneer factor, it has the capacity to remodel chromatin. We overexpressed this key master transcription factor but were unable to restore Ebf1 expression.

This illustrates that changes in local interactions are in fact the cause of abnormal gene expression as expression of PU.1, an upstream transcription factor cannot rescue the altered chromatin architecture or restore target gene expression.

Addback of Ebf1 cDNA rescues lymphoid lineage commitment and B cell colony formation in vitro and in vivo.

Hematopoietic differentiation requires dynamic 3-dimensional change. It is this very functionality which we investigate, going from locus specific loop-alterations to decreased accessibility, decreased expression, and finally to an observed defect in hematopoiesis

In this setting the pioneer factor PU.1 was unable to open chromatin at target sites in the absence of Stag2, however, B cell development/differentiation could be rescued through restoration of a downstream target gene beyond the choke point.

Many thanks to @rosslevinemd the best mentor in the biz and amazing colleagues @bowman_rl @vp_lavallee @dana_peer @job_dekker @dekker_lab and @RossLevineLab COMING SOON: The Viny Lab. Stay tuned! @cohesinlab

A huge debt of gratitude to @DamonRunyon @EdwardPEvansFdn and @theNCI for career development support—the war on cancer couldn’t have better allies! Also please welcome #EvansMDS to the #twitterverse! Follow them at @EdwardPEvansFdn