Michael B. Stadler

Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.

DNA-binding factors shape the mouse methylome at distal regulatory regions

Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.

Lineage-Specific Polycomb Targets and De Novo DNA Methylation Define Restriction and Potential of Neuronal Progenitors

Cellular differentiation entails loss of pluripotency and gain of lineage- and cell-type-specific characteristics. Using a murine system that progresses from stem cells to lineage-committed progenitors to terminally differentiated neurons, we analyzed DNA methylation and Polycomb-mediated histone H3 methylation (H3K27me3). We show that several hundred promoters, including pluripotency and germline-specific genes, become DNA methylated in lineage-committed progenitor cells, suggesting that DNA methylation may already repress pluripotency in progenitor cells. Conversely, we detect loss and acquisition of H3K27me3 at additional targets in both progenitor and terminal states. Surprisingly, many neuron-specific genes that become activated upon terminal differentiation are Polycomb targets only in progenitor cells. Moreover, promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation, suggesting context-dependent crosstalk between Polycomb and DNA methylation. These data suggest a model how de novo DNA methylation and dynamic switches in Polycomb targets restrict pluripotency and define the developmental potential of progenitor cells.

Encoding of conditioned fear in central amygdala inhibitory circuits

The central amygdala (CEA), a nucleus predominantly composed of GABAergic inhibitory neurons, is essential for fear conditioning. How the acquisition and expression of conditioned fear are encoded within CEA inhibitory circuits is not understood. Using in vivo electrophysiological, optogenetic and pharmacological approaches in mice, we show that neuronal activity in the lateral subdivision of the central amygdala (CEl) is required for fear acquisition, whereas conditioned fear responses are driven by output neurons in the medial subdivision (CEm). Functional circuit analysis revealed that inhibitory CEA microcircuits are highly organized and that cell-type-specific plasticity of phasic and tonic activity in the CEl to CEm pathway may gate fear expression and regulate fear generalization. Our results define the functional architecture of CEA microcircuits and their role in the acquisition and regulation of conditioned fear behaviour.

Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa

In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycomb-mediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.

Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs.

Adaptation to different levels of illumination is central to the function of the retina. Here, we demonstrate that levels of the miR-183/96/182 cluster, miR-204, and miR-211 are regulated by different light levels in the mouse retina. Concentrations of these microRNAs were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the respective changes. We identified the voltage-dependent glutamate transporter Slc1a1 as one of the miR-183/96/182 targets in photoreceptor cells. We found that microRNAs in retinal neurons decay much faster than microRNAs in nonneuronal cells. The high turnover is also characteristic of microRNAs in hippocampal and cortical neurons, and neurons differentiated from ES cells in vitro. Blocking activity reduced turnover of microRNAs in neuronal cells while stimulation with glutamate accelerated it. Our results demonstrate that microRNA metabolism in neurons is higher than in most other cells types and linked to neuronal activity.

Polycomb Group Proteins Ezh2 and Rnf2 Direct Genomic Contraction and Imprinted Repression in Early Mouse Embryos

Genomic imprinting regulates parental-specific expression of particular genes and is required for normal mammalian development. How imprinting is established during development is, however, largely unknown. To address this question, we studied the mouse Kcnq1 imprinted cluster at which paternal-specific silencing depends on expression of the noncoding RNA Kcnq1ot1. We show that Kcnq1ot1 is expressed from the zygote stage onward and rapidly associates with chromatin marked by Polycomb group (PcG) proteins and repressive histone modifications, forming a discrete repressive nuclear compartment devoid of RNA polymerase II, a configuration also observed at the Igf2r imprinted cluster. In this compartment, the paternal Kcnq1 cluster exists in a three-dimensionally contracted state. In vivo the PcG proteins Ezh2 and Rnf2 are independently required for genomic contraction and imprinted silencing. We propose that the formation of a parental-specific higher-order chromatin organization renders imprint clusters competent for monoallelic silencing and assign a central role to PcG proteins in this process.

The Transcriptome of Retinal Müller Glial Cells

Müller glial cells are the major type of glia in the mammalian retina. To identify the molecular machinery that defines Müller glial cell identity and function, single cell gene expression profiling was performed on Affymetrix microarrays. Identification of a cluster of genes expressed at high levels suggests a Müller glia core transcriptome, which likely underlies many of the functions of Müller glia. Expression of components of the cell cycle machinery and the Notch pathway, as well as of growth factors, chemokines, and lipoproteins might allow communication between Müller glial cells and the neurons that they support, including modulation of neuronal activity. This approach revealed a set of transcripts that were not previously characterized in (Müller) glia; validation of the expression of some of these genes was performed by in situ hybridization. Genes expressed exclusively by Müller glia were identified as novel markers. In addition, a novel BAC transgenic mouse that expresses Cre in Müller glia cells was generated. The molecular fingerprint of Müller glia provides a foundation for further studies of Müller glia development and function in normal and diseased states. J. Comp. Neurol. 509:225–238, 2008.

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN. Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C

Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non‐coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR‐214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well‐established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR‐214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR‐214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.