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The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints.