WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the ‘WIN’ site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks—if any—that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis.
Background Cohesin is an important structural regulator of the genome, regulating both three-dimensional genome organization and gene expression. The core cohesin trimer interacts with various HEAT repeat accessory subunits, yielding cohesin complexes of distinct compositions and potentially distinct functions. The roles of the two mutually exclusive HEAT repeat subunits PDS5A and PDS5B are not well understood. Results Here, we determine that PDS5A and PDS5B have highly similar localization patterns across the mouse embryonic stem cell (mESC) genome and they show a strong overlap with other cohesin HEAT repeat accessory subunits, STAG1 and STAG2. Using CRISPR/Cas9 genome editing to generate individual stable knockout lines for PDS5A and PDS5B, we find that loss of one PDS5 subunit does not alter the distribution of the other PDS5 subunit, nor the core cohesin complex. Both PDS5A and PDS5B are required for proper gene expression, yet they display only partially overlapping effects on gene targets. Remarkably, gene expression following dual depletion of the PDS5 HEAT repeat proteins does not completely overlap the gene expression changes caused by dual depletion of the STAG HEAT repeat proteins, despite the overlapping genomic distribution of all four proteins. Furthermore, dual loss of PDS5A and PDS5B decreases cohesin association with NIPBL and WAPL, reduces SMC3 acetylation, and does not alter overall levels of cohesin on the genome. Conclusions This work reveals the importance of PDS5A and PDS5B for proper cohesin function. Loss of either subunit has little effect on cohesin localization across the genome yet PDS5A and PDS5B are differentially required for gene expression.
Background The cohesin complex is essential for proper chromosome structure and gene expression. Defects in cohesin subunits and regulators cause changes in cohesin complex dynamics and thereby alter three-dimensional genome organization. However, the molecular mechanisms that drive cohesin localization and function remain poorly understood. Results In this study, we observe that loss of WIZ causes changes to cohesin localization that are distinct from loss of the known WIZ binding partner G9a. Whereas loss of WIZ uniformly increases cohesin levels on chromatin at known binding sites and leads to new, ectopic cohesin binding sites, loss of G9a does not. Ectopic cohesin binding on chromatin after the loss of WIZ occurs at regions that are enriched for activating histone modifications and transcription factors motifs. Furthermore, loss of WIZ causes changes in cohesin localization that are distinct from those observed by loss of WAPL, the canonical cohesin unloading factor. Conclusions The evidence presented here suggests that WIZ can function independently from its previously identified role with G9a and GLP in heterochromatin formation. Furthermore, while WIZ limits the levels and localization pattern of cohesin across the genome, it appears to function independently of WAPL-mediated cohesin unloading.
Sodium Voltage-Gated Channel Alpha Subunit 5 (SNC5A) plays a vital role in cardiac depolarization and has been implicated in Long QT Syndrome and sudden cardiac death. SCN5A encodes fetal and adult isoforms, differing by splicing of exon 6A or 6B, respectively. Fetal SCN5A decreases peak sodium current, leading to slower channel activation and inactivation and providing a potential substrate for arrhythmia. Little is known about developmental expression of these isoforms. We investigated developmental expression of SCN5A isoforms in infants, toddlers, and adults, including expression ratios in SUID cases and controls (0-12 months). We extracted RNA from heart tissue collected at autopsy (fetus-24 months) with 80 SUID cases and 27 controls, and 5 adult donor hearts. RNA Integrity Numbers ranged from 4.8-9.5. Relative SCN5A isoform expression was quantified by qRT-PCR. Non-parametric t-test and ANOVA were used; P<0.05 was considered statistically significant. Our results show a significant stepwise increase in adult/fetal isoform expression with age. Relative expression of adult/fetal SCN5A increased significantly from fetal-4 months to 5-24 months (P<0.0001), and again in adulthood (P<0.0001). There was no significant difference between case and control groups at 0-4 or 0-12 months. Expression of adult/fetal SCN5A increases significantly from birth to 24 months of age and into adulthood. Relative adult/fetal SCN5A expression is lowest during the neonatal period through 4 months, corresponding with the peak incidence of SUID. Further investigation of protein expression of SCN5A isoforms may shed light onto the age-dependent incidence of sudden death.
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