Charles C. Bell

X-chromosome inactivation (XCI) enables gene dosage compensation in XX eutherians. Long interspersed element-1 (LINE-1 or L1) retrotransposons are unusually abundant on the human X chromosome and are hypothesized to facilitate XCI. Here, we used long-read DNA sequencing to conduct a haplotype-aware analysis of engineered L1 integration preferences in the PA-1 human embryonic carcinoma cell line. Crucially, clonal XCI in PA-1 cells enabled derivation of active (Xa) and inactive (Xi) X-chromosome haplotypes. L1 integration strongly favored the Xi and other genomic regions that undergo DNA replication late in S-phase. These results suggest that the X chromosome is L1 rich because of XCI and imply that L1 integration preference for the Xi in XX individuals could potentially double the frequency of X-linked pathogenic L1 mutations in their XY descendants.

DOT1L and Menin are essential cofactors for the oncogenic activity of MLL fusion proteins (MLL-FPs) in leukaemia. However, the mechanisms underpinning the therapeutic effects of their inhibitors remain unclear. Here we identify a critical role for the non-canonical Polycomb repressive complex 1.1 (PRC1.1) in mediating the cellular responses to DOT1L and Menin inhibitors. Menin inhibition induces PRC1.1-dependent deposition of H2AK119ub to silence a subset of MLL-FP targets, whereas DOT1L inhibition results in a genome-wide increase in H2AK119ub. We show that enhanced PRC1.1 activity arises specifically from the progressive loss of DOT1L-mediated H3K79 methylation, independent of MLL-FP displacement or transcriptional repression. This regulatory crosstalk is conserved across cell types and is driven by direct biochemical antagonism between H3K79 methylation and PRC1 activity. Together, our findings establish DOT1L as a component of transcriptional memory co-opted in leukaemia and suggest it serves as the missing link balancing the opposing forces of the MLL–Polycomb axis.

Cell types are traditionally thought to be specified and stabilized by gene regulatory networks. Here, we explore how chromatin memory contributes to the specification and stabilization of cell states. Through pervasive, local, feedback loops, chromatin memory enables cell states that were initially unstable to become stable. Deeper appreciation of this self-stabilizing role for chromatin broadens our perspective of Waddington’s epigenetic landscape from a static surface with islands of stability shaped by evolution, to a plasticine surface molded by experience. With implications for the evolution of cell types, stabilization of resistant states in cancer, and the widespread plasticity of complex life.

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

Eukaryotic transcription factors (TFs) activate gene expression by recruiting cofactors to promoters. However, the relationships between TFs, promoters and their associated cofactors remain poorly understood. Here we combine GAL4-transactivation assays with comparative CRISPR–Cas9 screens to identify the cofactors used by nine different TFs and core promoters in human cells. Using this dataset, we associate TFs with cofactors, classify cofactors as ubiquitous or specific and discover transcriptional co-dependencies. Through a reductionistic, comparative approach, we demonstrate that TFs do not display discrete mechanisms of activation. Instead, each TF depends on a unique combination of cofactors, which influences distinct steps in transcription. By contrast, the influence of core promoters appears relatively discrete. Different promoter classes are constrained by either initiation or pause-release, which influences their dynamic range and compatibility with cofactors. Overall, our comparative cofactor screens characterize the interplay between TFs, cofactors and core promoters, identifying general principles by which they influence transcription.

Chromatin regulation involves the selective recruitment of chromatin factors to facilitate DNA repair, replication and transcription. Here we demonstrate the utility of coupling unbiased functional genomics with chromatin immunoprecipitation (CRISPR–ChIP) to identify the factors associated with active chromatin modifications in mammalian cells. Specifically, an integrated reporter containing a cis-regulatory element of interest and a single guide RNA provide a chromatinized template for a direct readout for regulators of histone modifications associated with actively transcribed genes such as H3K4me3 and H3K79me2. With CRISPR–ChIP, we identify all the nonredundant COMPASS complex members required for H3K4me3 and demonstrate that RNA polymerase II is dispensable for the maintenance of H3K4me3. As H3K79me2 has a putative oncogenic function in leukemia cells driven by MLL translocations, using CRISPR–ChIP we reveal a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex directed by the MLL fusion protein in a Menin-dependent manner and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10. Overall, CRISPR–ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR–Cas9 screens, we identify specific roles for MTF2–PRC2.1, PCGF1–PRC1.1 and Menin–KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin–KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

Transcription factors use DNA binding domains to recognise specific sequences and transactivation domains to recruit the cofactor proteins necessary for transcription. However, how specific cofactors contribute to transactivation at different genes remains unclear. Here, we couple Gal4-transactivation assays with comparative CRISPR-Cas9 screens to identify the cofactors required by nine different transcription factors and nine different core promoters in human cells. We classify cofactors as ubiquitous or specific, discover novel transcriptional co-dependencies and demonstrate that submodules within large co-activator complexes, such as the tail 2 and kinase modules of Mediator, facilitate transcriptional elongation. Rather than displaying discrete mechanisms of action, we discover that each TF requires a unique combination of cofactors, which influence its ability to potentiate distinct steps in the transcriptional process. Our findings help reconcile models of cofactor-promoter compatibility by demonstrating that transcription at different classes of promoters is constrained by either initiation or pause release. These differences dictate cofactor compatibility and the dynamic range of gene expression. Overall, our screens provide insight into TF-cofactor relationships and their ability to potentiate different steps in transcription at different classes of promoters.

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that are enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several activator protein-1 transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.

B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.