Asifa Akhtar

Dosage compensation (DC) in Drosophila equalizes X chromosome-linked gene expression via the male-specific lethal complex, which acetylates histone 4 lysine-16 on the male X chromosome. How DC is established during early embryogenesis remains obscure, largely due to the difficulty of obtaining sufficient sex-specific material at early embryonic stages. Here, we developed a high-throughput embryo sorting strategy and combined newly synthesized RNA-sequencing and RNA polymerase II (Pol II) profiling to investigate the mechanisms underlying the onset of DC in vivo. We found that changes in transcription initiation occur before detectable differences in transcriptional pausing or elongation and represent the primary determinant of enhanced mRNA output during DC establishment. It had hitherto been unclear whether DC establishment and maintenance exerted similar or distinct effects on transcriptional kinetics. Extending genome-wide profiling to S2 cells, we uncovered Pol II initiation as a general principle underlying both inception and perpetuation of DC-driven transcriptional up-regulation.

Bromodomain (BD) and extra-terminal domain (BET) proteins are key regulators of RNA polymerase II (Pol II)-mediated transcription and their BDs represent promising drug targets. Yet, the interplay between histone acetylation and the chromatin dynamics of individual BET proteins with respect to transcriptional regulation is not fully understood. Here in mouse embryonic stem cells, we uncover an essential role of BRD2 in maintaining Pol II recruitment at promoters through its interaction with TFIID, which becomes particularly critical under the conditions of impaired pause release. Combining rapid protein degradation, chemogenomics and super-resolution microscopy, we show that MOF-mediated histone H4 acetylation promotes BRD2 chromatin association, which in turn enables BRD2 clustering. Accordingly, MOF depletion or deletion of the BRD2’s intrinsically disordered region largely recapitulates defects in promoter enrichment and clustering of the transcription machinery observed upon BRD2 loss. Thus, these findings support a model in which histone acetylation-dependent spatiotemporal dynamics of BRD2 coordinate the transcription machinery to regulate transcription initiation.

Neural progenitor cells exhibit developmental plasticity as they can commit to distinct developmental trajectories. The male-specific lethal complex (MSLc) is linked to multiple developmental disorders, suggesting a role in neural fate commitment. To dissect MSLc function, we used a multipronged approach combining chronic and acute depletion models. Knockout of the MSLc scaffolding component MSL1 caused embryonic lethality by E10.5 (embryonic day 10.5), and single-cell multiomics revealed altered cell population composition across multiple germ layer–derived lineages, including neuroectoderm. Two-dimensional directed differentiation models showed that the MSLc facilitates accessibility at regulatory elements during early stages of neurogenesis. Neurodevelopmental genes displayed reduced enhancer-promoter contacts and failed to reach appropriate expression levels when the MSLc was absent early in neural differentiation. In contrast, MSLc loss at later stages did not recapitulate this phenotype, indicating that MSLc-mediated gene priming is a key mechanism enabling timely activation of lineage-specifying transcriptional programs.

KANSL2 is a subunit of the non-specific lethal (NSL) chromatin-modifying complex associated with glioblastoma (GBM) progression, but the intrinsic role of KANSL2 in GBM cells is poorly understood. By analyzing TCGA-GBM and GTEx datasets, we found that KANSL2 is upregulated in GBM and positively correlates with genes involved in ribosome biogenesis. Immunofluorescence and cell cycle analyses reveal a dynamic nuclear distribution, with KANSL2 becoming enriched in nucleoli mainly during G1/early S and G2 phases. Overexpression of KANSL2 increases 45S pre-rRNA and 28S rRNA levels, whereas its silencing reduces rRNA expression and histone H4 acetylation at lysines 5 and 8 within rDNA promoters. RNA-seq of patient-derived GBM spheroids confirms a global downregulation of ribosome biogenesis genes upon silencing of KANSL2. Together, these findings identify KANSL2 as a nuclear factor that transiently associates with nucleoli to promote rRNA transcription and ribosome biogenesis, supporting the biosynthetic and proliferative capacity of glioblastoma cells.

Liver disease is a leading cause of mortality worldwide. Emerging evidence highlights the significant role of epigenetic regulation in sustaining liver homeostasis, providing new therapeutic strategies for liver disease. Hepatocyte-specific deletion of the epigenetic regulator KANSL3, a key component of the NSL complex, results in early-onset liver disease marked by biliary hyperplasia and hepatic fibrosis. KANSL3 is essential for regulating hepatocyte transcriptional networks important for hepatic steroid and lipid metabolism through histone acetylation. Moreover, single-cell RNA sequencing demonstrated that the loss of KANSL3 disrupts the differentiation of hepatocytes in vivo. The transcriptional programs necessary for hepatocyte differentiation of ductal and fetal liver organoids were severely compromised in the absence of KANSL3. These findings collectively demonstrate a crucial role of the epigenetic regulator KANSL3 in hepatocyte differentiation in liver development and disease.

In mammals, chromosome-wide regulatory mechanisms ensure a balance of X-linked gene dosage between males (XY) and females (XX). In female cells, expression of genes from one of the two X chromosomes is curtailed, with selective accumulation of Xist-RNA, Xist-associated proteins, specific histone modifications (for example, H3K27me3) and Barr body formation observed throughout interphase. Here we show, using chromosome flow-sorting, that during mitosis, Xist-associated proteins dissociate from inactive X (Xi) chromosomes, while high levels of H3K27me3 and increased compaction of the Xi relative to active X (Xa), are retained. Proteomic comparison of mitotic Xi and Xa revealed that components of Hbo1 and Msl/Mof histone acetyltransferase complexes are significantly enriched on Xa as compared to Xi and autosomes. By contrast, inhibitors of histone acetylation co-enrich with Xi. Furthermore, inhibition of Hbo1 or deletion of Msl/Mof components functionally abolishes mitotic differences in H3K27me3 marking and chromosome compaction. These data uncover critical roles for acetylation pathways in preserving X chromosome properties during mitosis.

In mammals, chromosome-wide regulatory mechanisms ensure a balance of X-linked gene dosage between males (XY) and females (XX). In female cells, expression of genes from one of the two X-chromosomes is curtailed, with selective accumulation of Xist-RNA, Xist-associated proteins, specific histone modifications (eg. H3K27me3) and Barr body formation observed throughout interphase. Using chromosome flow-sorting, we show that during mitosis, Xist-associated proteins dissociate from inactive X (Xi) chromosomes, while high levels of H3K27me3 and increased compaction of the Xi relative to active X (Xa), are retained. Proteomic comparison of mitotic Xi and Xa revealed, unexpectedly, that components of Hbo1 and Msl/Mof histone acetyltransferase complexes co-enrich with Xa, while inhibitors of histone acetylation co-enrich with Xi. Furthermore, inhibition of Hbo1 or deletion of Msl/Mof components functionally abolishes mitotic differences in H3K27me3 marking and chromosome compaction. These data uncover critical roles for acetylation pathways in preserving X chromosome properties during mitosis.