Biology subjects

Greenberg, M. V. C.

Publications and source records attributed to Greenberg, M. V. C..

4 recordsLinked to original sources

bioRxiv2023

The embryonic DNA methylation program modulates the cis-regulatory landscape via CTCF antagonism

During mammalian embryogenesis, both the 5-cytosine DNA methylation (5meC) landscape and three-dimensional (3D) chromatin architecture are profoundly remodeled during a process known as "epigenetic reprogramming." An understudied aspect of epigenetic reprogramming is how the 5meC flux, per se, affects the 3D genome. This is pertinent given the 5meC-sensitivity of DNA binding for a key regulator of chromosome folding: CTCF. We profiled the CTCF binding landscape using a mouse embryonic stem cell (ESC) differentiation protocol that models the exit of naive pluripotency, wherein global DNA methylation levels start low and increase to somatic levels within four days. We took advantage of the fact that mouse ESCs lacking DNA methylation machinery exhibit globally similar differentiation dynamics, thus allowing for dissection of more subtle effects of CTCF misregulation on gene expression. We carried this out by performing CTCF HiChIP in both wild-type and mutant conditions to assess aberrant CTCF-CTCF contacts in the absence of 5meC. We went on to assess the impact that misregulated CTCF binding has on cis-regulatory contacts by performing H3K27ac HiChIP, given that H3K27ac is enriched on active promoters and enhancers. Using DNA methylation epigenome editing, we were able to directly demonstrate that the DNA methyl-mark is able to impact CTCF binding. Finally, a detailed dissection of the imprinted Zdbf2 gene showed how 5meC-antagonism of CTCF allows for proper gene regulation during differentiation. This work provides a comprehensive overview of how DNA methylation impacts the 3D genome in a relevant model for early embryonic events.

molecular biology↗

bioRxiv2023

DNA methylation shapes the Polycomb landscape during the exit from naïve pluripotency

In mammals, 5 methyl-cytosine (5mC) and Polycomb Repressive Complex 2 (PRC2)-deposited histone 3 lysine 27 trimethylation (H3K27me3) are generally mutually exclusive at CpG-rich regions. As mouse embryonic stem cells exit the naive pluripotent state, there is a massive gain of 5mC coincident with a restriction of broad H3K27me3 to 5mC-free, CpG-rich regions. To formally assess how 5mC shapes the H3K27me3 landscape, we profiled the epigenome of naive and differentiated cells in the presence and absence of the DNA methylation machinery. Surprisingly, we found that 5mC accumulation is not required to restrict most H3K27me3 domains. We went on to show that this 5mC-independent H3K27me3 restriction is mediated by aberrant expression of the PRC2 antagonist Ezhip. At the regions where 5mC appears to genuinely supplant H3K27me3, we identified 68 candidate genes that appeared to require 5mC deposition and/or H3K27me3 depletion for their activation in differentiated cells. Employing site-directed epigenome editing to directly modulate 5mC levels, we demonstrated that 5mC deposition is sufficient to antagonize H3K27me3 deposition and confer gene activation at individual candidates. Altogether, we systematically measured the antagonistic interplay between 5mC and H3K27me3 in a system that recapitulates early embryonic dynamics. Our results suggest that H3K27me3 restraint depends on 5mC, both directly and indirectly. This study also reveals a non-canonical role of 5mC in gene activation, which may be important not only for normal development but also for cancer progression, as oncogenic cells frequently exhibit dynamic replacement of 5mC for H3K27me3 and vice versa.

molecular biology↗

bioRxiv2022

DNA methylation restricts coordinated germline and neural fates in embryonic stem cell differentiation

Somatic DNA methylation is established early during mammalian development, as embryonic cells transition from naive to primed pluripotency. This precedes the emergence of the three somatic germ layers, but also the segregation of the germline that undergoes genome-wide DNA demethylation after specification. While DNA methylation is essential for embryogenesis, the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Using culture modeling of cellular transitions, we found that DNA methylation-free embryonic stem cells (ESCs) with a triple DNA methyltransferase knockout (TKO) normally progressed through the continuum of pluripotency states, but demonstrated skewed differentiation abilities towards neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells (PGCLCs), even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that the neural and germline lineages are linked by a similar enhancer dynamics during priming, defined by common sets of methyl-sensitive transcription factors that fail to be decommissioned in absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of preferred differentiation route during early development.

developmental biology↗

bioRxiv2021

A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements, and higher-order chromatin structures. Expectedly, these chromatin marks are indispensable for mammalian development and alterations often lead to diseases such as cancer. Molecularly, epigenetic mechanisms rely on factors to establish patterns, interpret them into a transcriptional output, and maintain them across cell divisions. A global picture of these phenomena has started to emerge over the years, yet many of the molecular actors remain to be discovered. In this context, we have developed a reporter system sensitive to epigenetic perturbations to report on repressive pathways based on Dazl, which is normally repressed in mouse ES cells. We used this system for a genome-wide CRISPR knock-out screen, which yielded expected hits (DNMT1, UHRF1, MGA), as well as novel candidates. We prioritized the candidates by secondary screens, and led further experiments on 6 of them: ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D. Our results show that all 6 candidates regulate the expression of germline genes. In addition, we find that removal of ZBTB14, KDM5C, SPOP and MCM3AP led to similar transcriptional responses, including a reactivation of the 2-cell like cell (2CLC) signature. Therefore, our genetic screen has identified new regulators of key cellular states.

molecular biology↗