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Inhibition of transcription leads to rewiring of locus-specific chromatin proteomes

Genome Res

2020 Tsukiyama, Toshio; Cucinotta, Christine E

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we employed Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. Taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent-cell specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein-protein relationships and protein functions at the chromatin template.

Nucleus structure and dynamics

Mol Biol Cell

2020 Hatch, Emily M


Evolution: Heterochromatin Diversity in Early-Branching Land Plants

Curr Biol

2020 Henikoff, Steven; Ahmad, Kamran

Chromatin profiling of a liverwort genome reveals an epigenomic landscape where the major mark of developmental silencing in later-branching land plants and in animals also targets subsets of transposons in this early-branching land plant lineage.

Dynamic evolution of de novo DNA methyltransferases in rodent and primate genomes

Mol Biol Evol

2020 Malik, Harmit S; Molaro, Antoine

Transcriptional silencing of retrotransposons via DNA methylation is paramount for mammalian fertility and reproductive fitness. During germ cell development, most mammalian species utilize the de novo DNA methyltransferases DNMT3A and DNMT3B to establish DNA methylation patterns. However, many rodent species deploy a third enzyme, DNMT3C, to selectively methylate the promoters of young retrotransposon insertions in their germline. The evolutionary forces that shaped DNMT3C's unique function are unknown. Using a phylogenomic approach, we confirm here that Dnmt3C arose through a single duplication of Dnmt3B that occurred around 60Mya in the last common ancestor of muroid rodents. Importantly, we reveal that DNMT3C is composed of two independently evolving segments: the latter two-thirds has undergone recurrent gene conversion with Dnmt3B, whereas the N-terminus has instead evolved under strong diversifying selection. We hypothesize that positive selection of Dnmt3C is the result of an ongoing evolutionary arms race with young retrotransposon lineages in muroid genomes. Interestingly, although primates lack DNMT3C, we find that the N-terminus of DNMT3A has also evolved under diversifying selection. Thus, the N-termini of two independent de novo methylation enzymes have evolved under diversifying selection in rodents and primates. We hypothesize that repression of young retrotransposons might be driving the recurrent innovation of a functional domain in the N-termini on germline DNMT3s in mammals.

The MYCL and MXD1 transcription factors regulate the fitness of murine dendritic cells

Proc Natl Acad Sci U S A

2020 Eisenman, Robert N

We previously found that MYCL is required by a Batf3-dependent classical dendritic cell subset (cDC1) for optimal CD8 T cell priming, but the underlying mechanism has remained unclear. The MAX-binding proteins encompass a family of transcription factors with overlapping DNA-binding specificities, conferred by a C-terminal basic helix-loop-helix domain, which mediates heterodimerization. Thus, regulation of transcription by these factors is dependent on divergent N-terminal domains. The MYC family, including MYCL, has actions that are reciprocal to the MXD family, which is mediated through the recruitment of higher-order activator and repressor complexes, respectively. As potent proto-oncogenes, models of MYC family function have been largely derived from their activity at supraphysiological levels in tumor cell lines. MYC and MYCN have been studied extensively, but empirical analysis of MYCL function had been limited due to highly restricted, lineage-specific expression in vivo. Here we observed that Mycl is expressed in immature cDC1s but repressed on maturation, concomitant with Mxd1 induction in mature cDC1s. We hypothesized that MYCL and MXD1 regulate a shared, but reciprocal, transcriptional program during cDC1 maturation. In agreement, immature cDC1s in Mycl -/- -deficient mice exhibited reduced expression of genes that regulate core biosynthetic processes. Mature cDC1s from Mxd1 -/- mice exhibited impaired ability to inhibit the transcriptional signature otherwise supported by MYCL. The present study reveals LMYC and MXD1 as regulators of a transcriptional program that is modulated during the maturation of Batf3-dependent cDC1s.

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress

Nat Commun

2020 Biggins, Susan; Tsukiyama, Toshio; Cutler, Samuel R

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

Connect-seq to superimpose molecular on anatomical neural circuit maps

Proc Natl Acad Sci U S A

2020 Buck, Linda B.; Lee, Eun Jeong; Hanchate, Naresh; Kuang, Donghui; Basom, Ryan S; Kondoh, Kunio; Trapnell, Cole

The mouse brain contains about 75 million neurons interconnected in a vast array of neural circuits. The identities and functions of individual neuronal components of most circuits are undefined. Here we describe a method, termed "Connect-seq," which combines retrograde viral tracing and single-cell transcriptomics to uncover the molecular identities of upstream neurons in a specific circuit and the signaling molecules they use to communicate. Connect-seq can generate a molecular map that can be superimposed on a neuroanatomical map to permit molecular and genetic interrogation of how the neuronal components of a circuit control its function. Application of this method to hypothalamic neurons controlling physiological responses to fear and stress reveals subsets of upstream neurons that express diverse constellations of signaling molecules and can be distinguished by their anatomical locations.

CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T

Curr Biol

2020 Hsieh, Emily

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3 while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components, including a divergent CENP-T homolog, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN homologs in other independently derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

Recurrent SRSF2 mutations in MDS affect both splicing and NMD

Genes Dev

2020 Bradley, Robert K

Oncogenic mutations in the RNA splicing factors SRSF2, SF3B1, and U2AF1 are the most frequent class of mutations in myelodysplastic syndromes and are also common in clonal hematopoiesis, acute myeloid leukemia, chronic lymphocytic leukemia, and a variety of solid tumors. They cause genome-wide splicing alterations that affect important regulators of hematopoiesis. Several mRNA isoforms promoted by the various splicing factor mutants comprise a premature termination codon (PTC) and are therefore potential targets of nonsense-mediated mRNA decay (NMD). In light of the mechanistic relationship between splicing and NMD, we sought evidence for a specific role of mutant SRSF2 in NMD. We show that SRSF2 Pro95 hot spot mutations elicit enhanced mRNA decay, which is dependent on sequence-specific RNA binding and splicing. SRSF2 mutants enhance the deposition of exon junction complexes (EJCs) downstream from the PTC through RNA-mediated molecular interactions. This architecture then favors the association of key NMD factors to elicit mRNA decay. Gene-specific blocking of EJC deposition by antisense oligonucleotides circumvents aberrant NMD promoted by mutant SRSF2, restoring the expression of PTC-containing transcript. Our study uncovered critical effects of SRSF2 mutants in hematologic malignancies, reflecting the regulation at multiple levels of RNA metabolism, from splicing to decay.

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody


2020 Bloom, Jesse D; Dingens, Adam S

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 μg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.