Actin is a key regulator of RNA polymerase (pol) II transcription. In complex with specific hnRNPs, it has been proposed that actin functions to recruit pol II coactivators during the elongation of nascent transcripts. Here, we show by affinity chromatography, protein-protein interaction assays, and biochemical fractionation of nuclear extracts that the histone acetyltransferase (HAT) PCAF associates with actin and hnRNP U. PCAF and the nuclear actin-associated HAT activity detected in the DNase I-bound protein fraction could be released by disruption of the actin-hnRNP U complex. In addition, actin, hnRNP U, and PCAF were found to be associated with the Ser2/5-and Ser2-phosphorylated pol II carboxy-terminal domain construct. Chromatin and RNA immunoprecipitation assays demonstrated that actin, hnRNP U, and PCAF are present at the promoters and coding regions of constitutively expressed pol II genes and that they are associated with ribonucleoprotein complexes. Finally, disruption of the actin-hnRNP U interaction repressed bromouridine triphosphate incorporation in living cells, suggesting that actin and hnRNP U cooperate with PCAF in the regulation of pol II transcription elongation.Eukaryotic gene transcription requires dynamic alterations of chromatin structure that are mediated by chromatin remodeling complexes and histone modifying enzymes (23, 28).For transcription competence, histone acetylation allows the switch between repressive and permissive chromatin structures through direct effects on nucleosome stability and through establishment of binding sites for regulatory proteins. Considerable progress in understanding the role of histone acetylation came from the discovery that the Saccharomyces cerevisiae transcription coactivator GCN5 and, more recently, other yeast and metazoan transcription cofactors are histone acetyltransferases (HATs). Mammalian homologs of the yeast cofactor GCN5 include PCAF and GCN5L (2,27,32,33,35). PCAF and GCN5L are encoded by distinct genes, and their expressions are differential and complementary in various tissues (33,35). Moreover, GCN5L is essential for mouse development, whereas PCAF is dispensable (33, 34). Human GCN5L and PCAF form parts of distinct multiprotein HAT complexes, namely the PCAF complex (17), the TFTC complex (1), and the STAGA complex (12). While these mammalian HAT complexes are still incompletely characterized, they have related but not identical subunit compositions.PCAF/GCN5, together with the p300/CREB-binding protein, is among the best-studied transcriptional coactivators (11). PCAF has been proposed to facilitate long-distance transcriptional enhancement by direct association with enhancer sequences (7). PCAF is also known to acetylate free histones or nucleosomes, primarily on lysine 14 of histone H3 (28), and its requirement as coactivator or HAT has been demonstrated for myogenesis and nuclear receptor-mediated and growth factor-signaled activation (28). However, it is presently unclear whether PCAF is also involved in the maintenance of effic...

SUMMARY Cardiac stem/progenitor cells hold great potential for regenerative therapies; however, the mechanisms regulating their expansion and differentiation remain insufficiently defined. Here we show that Ldb1 is a central regulator of genome organization in cardiac progenitor cells, which is crucial for cardiac lineage differentiation and heart development. We demonstrate that Ldb1 binds to the key regulator of cardiac progenitors, Isl1, and protects it from degradation. Furthermore, the Isl1/Ldb1 complex promotes long-range enhancer-promoter interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes that play key roles in cardiac progenitor cell function and cardiovascular development. Importantly, the expression of these genes was downregulated upon Ldb1 depletion and Isl1/Ldb1 haplodeficiency. In conclusion, the Isl1/Ldb1 complex orchestrates a network for heart-specific transcriptional regulation and coordination in three-dimensional space during cardiogenesis.

Generation of widely differing and specialized cell types from a single totipotent zygote involves large-scale transcriptional changes and chromatin reorganization. Pioneer transcription factors play key roles in programming the epigenome and facilitating recruitment of additional regulatory factors during successive cell lineage specification and differentiation steps. Here we show that Isl1 acts as a pioneer factor driving cardiomyocyte lineage commitment by shaping the chromatin landscape of cardiac progenitor cells. Using an Isl1 hypomorphic mouse line which shows congenital heart defects, genome-wide profiling of Isl1 binding together with RNA- and ATAC-sequencing of cardiac progenitor cells and their derivatives, we uncover a regulatory network downstream of Isl1 that orchestrates cardiogenesis. Mechanistically, we show that Isl1 binds to compacted chromatin and works in concert with the Brg1-Baf60c-based SWI/SNF complex to promote permissive cardiac lineage-specific alterations in the chromatin landscape not only of genes with critical functions in cardiac progenitor cells, but also of cardiomyocyte structural genes that are highly expressed when Isl1 itself is no longer present. Thus, the Isl1/Brg1-Baf60c complex plays a crucial role in orchestrating proper cardiogenesis and in establishing epigenetic memory of cardiomyocyte fate commitment.

Highlights d MYOD drives the re-wiring of chromatin interactions during trans-differentiation d MYOD alters chromatin interactions between cis-regulatory elements d MYOD re-wires insulated neighborhoods, hotspots of differential interactions d MYOD re-wiring of chromatin interactions temporally precedes transcriptional changes