Stormy J. Chamberlain

The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation.

PcG proteins mediate heritable transcriptional silencing by generating and recognizing covalent histone modifications. One conserved PcG complex, PRC2, is composed of several proteins including the histone methyltransferase (HMTase) Ezh2, the WD-repeat protein Eed, and the Zn-finger protein Suz12. Ezh2 methylates histone H3 on lysine 27 (H3K27), which serves as an epigenetic mark mediating silencing. H3K27 can be mono-, di-, or trimethylated (1mH3K27, 2mH3K27, and 3mH3K27, respectively). Hence, either PRC2 must be regulated so as to add one methyl group to certain nucleosomes but two or three to others, or distinct complexes must be responsible for 1m-, 2m-, and 3mH3K27. Consistent with the latter possibility, 2mH3K27 and 3mH3K27, but not 1mH3K27, are absent in Suz12-/- embryos, which lack both Suz12 and Ezh2 protein. Mammalian proteins required for 1mH3K27 have not been identified. Here, we demonstrate that unlike Suz12 and Ezh2, Eed is required not only for 2m- and 3mH3K27 but also global 1mH3K27. These results provide a functionally important distinction between PRC2 complex components and implicate Eed in PRC2-independent histone methylation.

Polycomb Repressive Complex 2 Is Dispensable for Maintenance of Embryonic Stem Cell Pluripotency

Polycomb repressive complex 2 (PRC2) methylates histone H3 tails at lysine 27 and is essential for embryonic development. The three core components of PRC2, Eed, Ezh2, and Suz12, are also highly expressed in embryonic stem (ES) cells, where they are postulated to repress developmental regulators and thereby prevent differentiation to maintain the pluripotent state. We performed gene expression and chimera analyses on low‐ and high‐passage Eednull ES cells to determine whether PRC2 is required for the maintenance of pluripotency. We report here that although developmental regulators are overexpressed in Eednull ES cells, both low‐ and high‐passage cells are functionally pluripotent. We hypothesize that they are pluripotent because they maintain expression of critical pluripotency factors. Given that EED is required for stability of EZH2, the catalytic subunit of the complex, these data suggest that PRC2 is not necessary for the maintenance of the pluripotent state in ES cells. We propose a positive‐only model of embryonic stem cell maintenance, where positive regulation of pluripotency factors is sufficient to mediate stem cell pluripotency.

Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader–Willi syndromes

Angelman syndrome (AS) and Prader–Willi syndrome (PWS) are neurodevelopmental disorders of genomic imprinting. AS results from loss of function of the ubiquitin protein ligase E3A (UBE3A) gene, whereas the genetic defect in PWS is unknown. Although induced pluripotent stem cells (iPSCs) provide invaluable models of human disease, nuclear reprogramming could limit the usefulness of iPSCs from patients who have AS and PWS should the genomic imprint marks be disturbed by the epigenetic reprogramming process. Our iPSCs derived from patients with AS and PWS show no evidence of DNA methylation imprint erasure at the cis-acting PSW imprinting center. Importantly, we find that, as in normal brain, imprinting of UBE3A is established during neuronal differentiation of AS iPSCs, with the paternal UBE3A allele repressed concomitant with up-regulation of the UBE3A antisense transcript. These iPSC models of genomic imprinting disorders will facilitate investigation of the AS and PWS disease processes and allow study of the developmental timing and mechanism of UBE3A repression in human neurons.

Topoisomerases facilitate transcription of long genes linked to autism

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

Neurodevelopmental disorders involving genomic imprinting at human chromosome 15q11-q13.

Human chromosome 15q11-q13 is subject to regulation by genomic imprinting, an epigenetic process by which genes are expressed in a parent-of-origin specific manner. Three neurodevelopmental disorders, Prader-Willi syndrome, Angelman syndrome, and 15q duplication syndrome, result from aberrant expression of imprinted genes in this region. Here, we review the current literature pertaining to mouse models and recently identified patients with atypical deletions, which shed light on the epigenetic regulation of the chromosome 15q11-q13 subregion and the genes that are responsible for the phenotypic outcomes of these disorders.

Angelman Syndrome, a Genomic Imprinting Disorder of the Brain

Harry Angelman, an English pediatrician, reported three cases of “Puppet Children” in 1965 ([Angelman, 1965][1]). These individuals displayed severe intellectual disability, ataxia, absent speech, jerky arm movements and bouts of inappropriate laughter. More cases were described as “Happy

Subtelomeric hotspots of aberrant 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC), which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hESCs, we find that iPSCs tend to form large-scale (100 kb–1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly, these 5hmC aberrant hotspots largely coincide (∼ 80%) with aberrant iPSC–ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC–hESC differences.

A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb10

Genomic imprinting is a developmental mechanism that mediates parent‐of‐origin‐specific expression in a subset of genes. How the tissue specificity of imprinted gene expression is controlled remains poorly understood. As a model to address this question, we studied Grb10, a gene that displays brain‐specific expression from the paternal chromosome. Here, we show in the mouse that the paternal promoter region is marked by allelic bivalent chromatin enriched in both H3K4me2 and H3K27me3, from early embryonic stages onwards. This is maintained in all somatic tissues, but brain. The bivalent domain is resolved upon neural commitment, during the developmental window in which paternal expression is activated. Our data indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain. This finding highlights a novel mechanism to control tissue‐specific imprinting.

Induced pluripotent stem (iPS) cells as in vitro models of human neurogenetic disorders

The recent discovery of genomic reprogramming of human somatic cells into induced pluripotent stem cells offers an innovative and relevant approach to the study of human genetic and neurogenetic diseases. By reprogramming somatic cells from patient samples, cell lines can be isolated that self-renew indefinitely and have the potential to develop into multiple different tissue lineages. Additionally, the rapid progress of research on human embryonic stem cells has led to the development of sophisticated in vitro differentiation protocols that closely mimic mammalian development. In particular, there have been significant advances in differentiating human pluripotent stem cells into defined neuronal types. Here, we summarize the experimental approaches employed in the rapidly evolving area of somatic cell reprogramming and the methodologies for differentiating human pluripotent cells into neurons. We also discuss how the availability of patient-specific fibroblasts offers a unique opportunity for studying and modeling the effects of specific gene defects on human neuronal development in vitro and for testing small molecules or other potential therapies for the relevant neurogenetic disorders.

Transcription is required to establish maternal imprinting at the Prader-Willi syndrome and Angelman syndrome locus.

The Prader-Willi syndrome (PWS [MIM 17620]) and Angelman syndrome (AS [MIM 105830]) locus is controlled by a bipartite imprinting center (IC) consisting of the PWS-IC and the AS-IC. The most widely accepted model of IC function proposes that the PWS-IC activates gene expression from the paternal allele, while the AS-IC acts to epigenetically inactivate the PWS-IC on the maternal allele, thus silencing the paternally expressed genes. Gene order and imprinting patterns at the PWS/AS locus are well conserved from human to mouse; however, a murine AS-IC has yet to be identified. We investigated a potential regulatory role for transcription from the Snrpn alternative upstream exons in silencing the maternal allele using a murine transgene containing Snrpn and three upstream exons. This transgene displayed appropriate imprinted expression and epigenetic marks, demonstrating the presence of a functional AS-IC. Transcription of the upstream exons from the endogenous locus correlates with imprint establishment in oocytes, and this upstream exon expression pattern was conserved on the transgene. A transgene bearing targeted deletions of each of the three upstream exons exhibited loss of imprinting upon maternal transmission. These results support a model in which transcription from the Snrpn upstream exons directs the maternal imprint at the PWS-IC.