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Summary: DNA methylation has been implicated as an epigenetic component of mechanisms that stabilize cell-fate decisions. Here, we have characterized the methylomes of human female hematopoietic stem/progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with lineage-specific genes were often methylated in the opposing lineage. In HSPCs, these sites tended to show intermediate, complex patterns that resolve to uniformity upon differentiation, by increased or decreased methylation. Promoter HMRs shared across diverse cell types typically display a constitutive core that expands and contracts in a lineage-specific manner to fine-tune the expression of associated genes. Many newly identified intergenic HMRs, both constitutive and lineage specific, were enriched for factor binding sites with an implied role in genome organization and regulation of gene expression, respectively. Overall, our studies represent an important reference data set and provide insights into directional changes in DNA methylation as cells adopt terminal fates. [ABSTRACT FROM AUTHOR]

Summary: During germ cell and preimplantation development, mammalian cells undergo nearly complete reprogramming of DNA methylation patterns. We profiled the methylomes of human and chimp sperm as a basis for comparison to methylation patterns of ESCs. Although the majority of promoters escape methylation in both ESCs and sperm, the corresponding hypomethylated regions show substantial structural differences. Repeat elements are heavily methylated in both germ and somatic cells; however, retrotransposons from several subfamilies evade methylation more effectively during male germ cell development, whereas other subfamilies show the opposite trend. Comparing methylomes of human and chimp sperm revealed a subset of differentially methylated promoters and strikingly divergent methylation in retrotransposon subfamilies, with an evolutionary impact that is apparent in the underlying genomic sequence. Thus, the features that determine DNA methylation patterns differ between male germ cells and somatic cells, and elements of these features have diverged between humans and chimpanzees. [Copyright &y& Elsevier]

Genetic models suggested that SMARCA5 was required for DNA-templated events including transcription, DNA replication, and DNA repair. We engineered a degron tag into the endogenous alleles of SMARCA5 , a catalytic component of the imitation switch complexes in three different human cell lines to define the effects of rapid degradation of this key regulator. Degradation of SMARCA5 was associated with a rapid increase in global nucleosome repeat length, which may allow greater chromatin compaction. However, there were few changes in nascent transcription within the first 6 h of degradation. Nevertheless, we demonstrated a requirement for SMARCA5 to control nucleosome repeat length at G 1 /S and during the S phase. SMARCA5 co-localized with CTCF and H2A.Z, and we found a rapid loss of CTCF DNA binding and disruption of nucleosomal phasing around CTCF binding sites. This spatiotemporal analysis indicates that SMARCA5 is continuously required for maintaining nucleosomal spacing. [Display omitted] • Target-specific protein degradation of endogenous human SMARCA5 • Degradation of SMARCA5 leads to a rapid loss in CTCF DNA binding • SMARCA5 regulates nucleosome repeat length independent of the cell cycle • CTCF and SMARCA5 co-localize at H2A.Z-containing sites Bomber et al. utilize degron tagging of the endogenous human chromatin-remodeling enzyme SMARCA5, coupled with a multi-omics approach, to define the requirements for SMARCA5-mediated nucleosome sliding. SMARCA5 co-localized with H2A.Z and CTCF and was required for CTCF DNA binding, nucleosomal phasing at these sites, and maintaining nucleosome repeat length. [ABSTRACT FROM AUTHOR]

Summary: Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5′ terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5′ terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5′ terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5′ end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine. [Copyright &y& Elsevier]

DNA methylation of enhancers is dynamic, cell-type specific, and vital for cell fate progression. However, current models inadequately define its role within the hierarchy of gene regulation. Analysis of independent datasets shows an unanticipated overlap between DNA methylation and chromatin accessibility at enhancers of steady-state stem cells, suggesting that these two opposing features might exist concurrently. To define their temporal relationship, we developed ATAC-Me, which probes accessibility and methylation from single DNA library preparations. We identified waves of accessibility occurring rapidly across thousands of myeloid enhancers in a monocyte-to-macrophage cell fate model. Prolonged methylation states were observed at a majority of these sites, while transcription of nearby genes tracked closely with accessibility. ATAC-Me uncovers a significant disconnect between chromatin accessibility, DNA methylation status, and gene activity. This unexpected observation highlights the value of ATAC-Me in constructing precise molecular timelines for understanding the role of DNA methylation in gene regulation. • ATAC-Me simultaneously measures chromatin accessibility and DNA methylation (DNAme) • Enhancer accessibility and DNAme change independently during cell fate transitions • Loss of DNAme is delayed in nascent open chromatin regions • Transcriptional changes track with new enhancer accessibility despite prolonged DNAme Barnett et al. describe ATAC-Me, an approach that captures chromatin accessibility and DNA methylation simultaneously from a single DNA fragment library. ATAC-Me was used to probe temporal relationships between epigenetic and gene regulatory changes at enhancers during myeloid differentiation of THP1 monocytes, revealing a decoupling of chromatin and DNA methylation changes at transitioning enhancers. [ABSTRACT FROM AUTHOR]

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: α, β, δ, and γ; when and how the Neurog3 + cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3 + cells co-expressing Myt1 (i.e., Myt1 + Neurog3 + ) were biased toward β cell fate, while those not simultaneously expressing Myt1 (Myt1 - Neurog3 + ) favored α fate. Myt1 manipulation only marginally affected α versus β cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1 + Neurog3 + cells displayed higher Dnmt1 expression and enhancer methylation at Arx, an α-fate-promoting gene. Inhibiting Dnmts in pancreatic progenitors promoted α cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored β cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs., (Published by Elsevier Inc.)

Pregnancy is the major modulator of mammary gland activity. It induces a tremendous expansion of the mammary epithelium and the generation of alveolar structures for milk production. Anecdotal evidence from multiparous humans indicates that the mammary gland may react less strongly to the first pregnancy than it does to subsequent pregnancies. Here, we verify that the mouse mammary gland responds more robustly to a second pregnancy, indicating that the gland retains a long-term memory of pregnancy. A comparison of genome-wide profiles of DNA methylation in isolated mammary cell types reveals substantial and long-lasting alterations. Since these alterations are maintained in the absence of the signal that induced them, we term them epigenetic. The majority of alterations in DNA methylation affect sites occupied by the Stat5a transcription factor and mark specific genes that are upregulated during pregnancy. We postulate that the epigenetic memory of a first pregnancy primes the activation of gene expression networks that promote mammary gland function in subsequent reproductive cycles. More broadly, our data indicate that physiological experience can broadly alter epigenetic states, functionally modifying the capacity of the affected cells to respond to later stimulatory events., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)