Dynamic Transcription of Distinct Classes of Endogenous Retroviral Elements Marks Specific Populations of Early Human Embryonic Cells

SummaryAbout half of the human genome consists of highly repetitive elements, most of which are considered dispensable for human life. Here, we report that repetitive elements originating from endogenous retroviruses (ERVs) are systematically transcribed during human early embryogenesis in a stage-specific manner. Our analysis highlights that the long terminal repeats (LTRs) of ERVs provide the template for stage-specific transcription initiation, thereby generating hundreds of co-expressed, ERV-derived RNAs. Conversion of human embryonic stem cells (hESCs) to an epiblast-like state activates blastocyst-specific ERV elements, indicating that their activity dynamically reacts to changes in regulatory networks. In addition to initiating stage-specific transcription, many ERV families contain preserved splice sites that join the ERV segment with non-ERV exons in their genomic vicinity. In summary, we find that ERV expression is a hallmark of cellular identity and cell potency that characterizes the cell populations in early human embryos

A Biophysical Model for Analysis of Transcription Factor Interaction and Binding Site Arrangement from Genome-Wide Binding Data

BACKGROUND:How transcription factors (TFs) interact with cis-regulatory sequences and interact with each other is a fundamental, but not well understood, aspect of gene regulation. METHODOLOGY/PRINCIPAL FINDINGS:We present a computational method to address this question, relying on the established biophysical principles. This method, STAP (sequence to affinity prediction), takes into account all combinations and configurations of strong and weak binding sites to analyze large scale transcription factor (TF)-DNA binding data to discover cooperative interactions among TFs, infer sequence rules of interaction and predict TF target genes in new conditions with no TF-DNA binding data. The distinctions between STAP and other statistical approaches for analyzing cis-regulatory sequences include the utility of physical principles and the treatment of the DNA binding data as quantitative representation of binding strengths. Applying this method to the ChIP-seq data of 12 TFs in mouse embryonic stem (ES) cells, we found that the strength of TF-DNA binding could be significantly modulated by cooperative interactions among TFs with adjacent binding sites. However, further analysis on five putatively interacting TF pairs suggests that such interactions may be relatively insensitive to the distance and orientation of binding sites. Testing a set of putative Nanog motifs, STAP showed that a novel Nanog motif could better explain the ChIP-seq data than previously published ones. We then experimentally tested and verified the new Nanog motif. A series of comparisons showed that STAP has more predictive power than several state-of-the-art methods for cis-regulatory sequence analysis. We took advantage of this power to study the evolution of TF-target relationship in Drosophila. By learning the TF-DNA interaction models from the ChIP-chip data of D. melanogaster (Mel) and applying them to the genome of D. pseudoobscura (Pse), we found that only about half of the sequences strongly bound by TFs in Mel have high binding affinities in Pse. We show that prediction of functional TF targets from ChIP-chip data can be improved by using the conservation of STAP predicted affinities as an additional filter. CONCLUSIONS/SIGNIFICANCE:STAP is an effective method to analyze binding site arrangements, TF cooperativity, and TF target genes from genome-wide TF-DNA binding data

p53 functions as a negative regulator of osteoblastogenesis, osteoblast-dependent osteoclastogenesis, and bone remodeling

p53 is a well known tumor suppressor. We show that p53 also regulates osteoblast differentiation, bone formation, and osteoblast-dependent osteoclast differentiation. Indeed, p53−/− mice display a high bone mass phenotype, and p53−/− osteoblasts show accelerated differentiation, secondary to an increase in expression of the osteoblast differentiation factor osterix, as a result. Reporter assays indicate that p53 represses osterix transcription by the minimal promoter in a DNA-binding–independent manner. In addition, p53−/− osteoblasts have an enhanced ability to favor osteoclast differentiation, in association with an increase in expression of macrophage-colony stimulating factor, which is under the control of osterix. Furthermore, inactivating p53 is sufficient to rescue the osteoblast differentiation defects observed in mice lacking c-Abl, a p53-interacting protein. Thus, these results identify p53 as a novel regulator of osteoblast differentiation, osteoblast-dependent osteoclastogenesis, and bone remodeling

Vestiges of a DNA methylation system in Drosophila melanogaster?

NON

Methylation of H3-Lysine 79 Is Mediated by a New Family of HMTases without a SET Domain

AbstractThe N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation [1]. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity [2–4]. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family [3]. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase

MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex

Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins (1–4). The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases (5–7). Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro4 and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex (1,9). An affinity-purified HDAC1 corepressor complex (10,11) also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylationdependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes

Reduced Oct4 expression directs a robust pluripotent state with distinct signaling activity and increased enhancer occupancy by Oct4 and Nanog

10.1016/j.stem.2013.04.023Cell Stem Cell125531-54

Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson\u27s disease modeling

Recent studies have demonstrated the generation of midbrain-like organoids (MOs) from human pluripotent stem cells. However, the low efficiency of MO generation and the relatively immature and heterogeneous structures of the MOs hinder the translation of these organoids from the bench to the clinic. Here we describe the robust generation of MOs with homogeneous distribution of midbrain dopaminergic (mDA) neurons. Our MOs contain not only mDA neurons but also other neuronal subtypes as well as functional glial cells including astrocytes and oligodendrocytes. Furthermore, our MOs exhibit mDA neuron-specific cell death upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, indicating that MOs could be a proper human model system for studying the in vivo pathology of Parkinson\u27s disease (PD). Our optimized conditions for producing homogeneous and mature MOs might provide an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD

Lewy Body–like Inclusions in Human Midbrain Organoids Carrying Glucocerebrosidase and α‐Synuclein Mutations

ObjectiveWe utilized human midbrain-like organoids (hMLOs) generated from human pluripotent stem cells carrying glucocerebrosidase gene (GBA1) and α-synuclein (α-syn; SNCA) perturbations to investigate genotype-to-phenotype relationships in Parkinson disease, with the particular aim of recapitulating α-syn– and Lewy body–related pathologies and the process of neurodegeneration in the hMLO model.MethodsWe generated and characterized hMLOs from GBA1−/− and SNCA overexpressing isogenic embryonic stem cells and also generated Lewy body–like inclusions in GBA1/SNCA dual perturbation hMLOs and conduritol-b-epoxide–treated SNCA triplication hMLOs.ResultsWe identified for the first time that the loss of glucocerebrosidase, coupled with wild-type α-syn overexpression, results in a substantial accumulation of detergent-resistant, β-sheet–rich α-syn aggregates and Lewy body–like inclusions in hMLOs. These Lewy body–like inclusions exhibit a spherically symmetric morphology with an eosinophilic core, containing α-syn with ubiquitin, and can also be formed in Parkinson disease patient–derived hMLOs. We also demonstrate that impaired glucocerebrosidase function promotes the formation of Lewy body–like inclusions in hMLOs derived from patients carrying the SNCA triplication.InterpretationTaken together, the data indicate that our hMLOs harboring 2 major risk factors (glucocerebrosidase deficiency and wild-type α-syn overproduction) of Parkinson disease provide a tractable model to further elucidate the underlying mechanisms for progressive Lewy body formation. ANN NEUROL 2021;90:490–50