Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers.

DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic ß-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3+ embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and ß-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and ß-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for ß-cell reprogramming in diabetes and diagnosis of ß-cell death using methylation patterns of circulating DNA.

Annotating the genome by DNA methylation.

DNA methylation plays a prominent role in setting up and stabilizing the molecular design of gene regulation and by understanding this process one gains profound insight into the underlying biology of mammals. In this article, we trace the discoveries that provided the foundations of this field, starting with the mapping of methyl groups in the genome and the experiments that helped clarify how methylation patterns are maintained through cell division. We then address the basic relationship between methyl groups and gene repression, as well as the molecular rules involved in controlling this process during development in vivo. Finally, we describe ongoing work aimed at defining the role of this modification in disease and deciphering how it may serve as a mechanism for sensing the environment.

Tocotrienol Treatment in Familial Dysautonomia: Open-Label Pilot Study.

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy, primarily presented in Ashkenazi Jews. The most common mutation in FD patients results from a single base pair substitution of an intronic splice site in the IKBKAP gene which disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP). To date, treatment of FD patients remains preventative, symptomatic and supportive. Based on previous in vitro evidence that tocotrienols, members of the vitamin E family, upregulate transcription of the IKBKAP gene, we aimed to investigate whether a similar effects was observed in vivo. In the current study, we assessed the effects of tocotrienol treatment on FD patients' symptoms and IKBKAP expression in white blood cells. The initial daily doses of 50 or 100 mg tocotrienol, doubled after 3 months, was administered to 32 FD patients. Twenty-eight FD patients completed the 6-month study. The first 3 months of tocotrienol treatment was associated with a significant increase in IKBKAP expression level in FD patients' blood. Despite doubling the dose after the initial 3 months of treatment, IKBKAP expression level returned to baseline by the end of the 6-month treatment. Clinical improvement was noted in the reported clinical questionnaire (with regard to dizziness, bloching, sweating, number of pneumonia, cough episodes, and walking stability), however, no significant effect was observed in any clinical measurements (weight, height, oxygen saturation, blood pressure, tear production, histamine test, vibration threshold test, nerve conduction, and heart rate variability) following Tocotrienol treatment. In conclusion, tocotrienol treatment appears significantly beneficial by clinical evaluation for some FD patients in a few clinical parameters; however it was not significant by clinical measurements. This open-label study shows the complexity of effect of tocotrienol treatment on FD patients' clinical outcomes and on IKBKAP expression level compared to in vitro results. A longitudinal study with an increased sample size is required in the future to better understand tocotrienol affect on FD patients.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation.

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.

IKAP deficiency in an FD mouse model and in oligodendrocyte precursor cells results in downregulation of genes involved in oligodendrocyte differentiation and myelin formation.

The splice site mutation in the IKBKAP gene coding for IKAP protein leads to the tissue-specific skipping of exon 20, with concomitant reduction in IKAP protein production. This causes the neurodevelopmental, autosomal-recessive genetic disorder - Familial Dysautonomia (FD). The molecular hallmark of FD is the severe reduction of IKAP protein in the nervous system that is believed to be the main reason for the devastating symptoms of this disease. Our recent studies showed that in the brain of two FD patients, genes linked to oligodendrocyte differentiation and/or myelin formation are significantly downregulated, implicating IKAP in the process of myelination. However, due to the scarcity of FD patient tissues, these results awaited further validation in other models. Recently, two FD mouse models that faithfully recapitulate FD were generated, with two types of mutations resulting in severely low levels of IKAP expression. Here we demonstrate that IKAP deficiency in these FD mouse models affects a similar set of genes as in FD patients' brains. In addition, we identified two new IKAP target genes involved in oligodendrocyte cells differentiation and myelination, further underscoring the essential role of IKAP in this process. We also provide proof that IKAP expression is needed cell-autonomously for the regulation of expression of genes involved in myelin formation since knockdown of IKAP in the Oli-neu oligodendrocyte precursor cell line results in similar deficiencies. Further analyses of these two experimental models will compensate for the lack of human postmortem tissues and will advance our understanding of the role of IKAP in myelination and the disease pathology.

Dynamic changes in IKBKAP mRNA levels during crisis of familial dysautonomia patients.

UNLABELLED: Familial dysautonomia is a neurodegenerative, genetic disorder caused by an autosomal recessive mutation in the IKBKAP gene, which encodes the IkB kinase complex-associated protein. Familial dysautonomia patients have recurrent crises characterized by bouts of nausea, vomiting, hypertension, tachycardia, sweating, blotching and personality changes. The dysautonomia crisis is usually triggered by stressful physiological or emotional events, however the pathophysiology of the crisis is not yet fully clear and little is known about the molecular mechanisms involved in onset and consequences of the crisis. OBJECTIVE: We have investigated the dysautonomia crisis by evaluating the expression of the familial dysautonomia gene - IKBKAP, in patients during different crisis stages. METHOD: Baseline IKBKAP mRNA levels in white blood cells were evaluated in thirteen FD patients (fourteen crisis events) and compared to mRNA levels at the onset, during, and after recovery from the crisis. RESULTS: We have found a significant decrease in IKBKAP mRNA level during the crisis, which is restored to a baseline level after recovery from the crisis. CONCLUSION: We speculate that the familial dysautonomia crisis pathophysiology might be related, at least in part, to the down regulation of the IKBKAP gene. Yet, it is still unclear whether the down regulation in IKBKAP mRNA is caused by the physiological stress events which have triggered the crisis or whether this molecular change is a consequence of the crisis.

Mechanisms of activation of the paternally expressed genes by the Prader-Willi imprinting center in the Prader-Willi/Angelman syndromes domains.

The Prader-Willi syndrome/Angelman syndrome (PWS/AS) imprinted domain is regulated by a bipartite imprinting control center (IC) composed of a sequence around the SNRPN promoter (PWS-IC) and a 880-bp sequence located 35 kb upstream (AS-IC). The AS-IC imprint is established during gametogenesis and confers repression upon PWS-IC on the maternal allele. Mutation at PWS-IC on the paternal allele leads to gene silencing across the entire PWS/AS domain. This silencing implies that PWS-IC functions on the paternal allele as a bidirectional activator. Here we examine the mechanism by which PWS-IC activates the paternally expressed genes (PEGs) using transgenes that include the PWS-IC sequence in the presence or absence of AS-IC and NDN, an upstream PEG, as an experimental model. We demonstrate that PWS-IC is in fact an activator of NDN. This activation requires an unmethylated PWS-IC in the gametes and during early embryogenesis. PWS-IC is dispensable later in development. Interestingly, a similar activation of a nonimprinted gene (APOA1) was observed, implying that PWS-IC is a universal activator. To decipher the mechanism by which PWS-IC confers activation of remote genes, we performed methylated DNA immunoprecipitation (MeDIP) array analysis on lymphoblast cell lines that revealed dispersed, rather than continued differential methylation. However, chromatin conformation capture (3c) experiments revealed a physical interaction between PWS-IC and the PEGs, suggesting that activation of PEGs may require their proximity to PWS-IC.

IKAP/Elp1 involvement in cytoskeleton regulation and implication for familial dysautonomia.

Deficiency in the IKAP/Elp1 protein leads to the recessive sensory autosomal congenital neuropathy which is called familial dysautonomia (FD). This protein was originally identified as a role player in transcriptional elongation being a subunit of the RNAPII transcriptional Elongator multi-protein complex. Subsequently, IKAP/Elp1 was shown to play various functions in the cytoplasm. Here, we describe experiments performed with IKAP/Elp1 downregulated cell lines and FD-derived cells and tissues. Immunostaining of the cytoskeleton component α-tubulin in IKAP/Elp1 downregulated cells revealed disorganization of the microtubules (MTs) that was reflected in aberrant cell shape and process formation. In contrast to a recent report on the decrease in α-tubulin acetylation in IKAP/Elp1 downregulated cells, we were unable to observe any effect of IKAP/Elp1 deficiency on α-tubulin acetylation in the FD cerebrum and in a variety of IKAP/Elp1 downregulated cell lines. To explore possible candidates involved in the observed aberrations in MTs, we focused on superior cervical ganglion-10 protein (SCG10), also called STMN2, which is known to be an MT destabilizing protein. We have found that SCG10 is upregulated in the IKAP/Elp1-deficient FD cerebrum, FD fibroblasts and in IKAP/Elp1 downregulated neuroblastoma cell line. To better understand the effect of IKAP/Elp1 deficiency on SCG10 expression, we investigated the possible involvement of RE-1-silencing transcription factor (REST), a known repressor of the SCG10 gene. Indeed, REST was downregulated in the IKAP/Elp1-deficient FD cerebrum and IKAP/Elp1 downregulated neuroblastoma cell line. These results could shed light on a possible link between IKAP/Elp1 deficiency and cytoskeleton destabilization.

Protein-binding elements establish in the oocyte the primary imprint of the Prader-Willi/Angelman syndromes domain.

Imprinting of the PWS/AS 2.4 Mb domain in the human is controlled by a paternally active imprinting center (PWS-IC). PWS-IC on the maternal allele is methylated and inactivated by an 880-bp sequence (AS-IC) located 30 kb upstream. In this communication, we report the identification of 7 cis acting elements within AS-IC. The elements: DMR, DNS, 2 OCTA sequences, SOX, E1, and E2 bind specific proteins that form at least 2 protein complexes. Using variants of an imprinted transgene, mutated at the elements each at a time, we show that (i) all 7 elements are involved in the methylation and inactivation of the maternal PWS-IC; (ii) the OCTA and SOX elements that bind a protein complex, and the E1 and E2 elements, function in establishing the primary imprint that constitutes an active and unmethylated AS-IC in the oocyte; (iii) DNS and DMR bind a multiprotein complex that may facilitate interaction between AS-IC and PWS-IC, mediating the inactivation in cis of PWS-IC; and (iv) all 7 elements participate in maintaining an unmethylated PWS-IC in the oocyte, which is essential for its maternal methylation later in development. Altogether, the above observations imply that the cis acting elements on AS-IC display diverse functions in establishing the imprints at both AS-IC and PWS-IC in the oocyte. A postulated epigenetic mark imprints the PWS-IC in the oocyte and maintains its inactive status during development before it is translated into maternal methylation.

MeCP2 involvement in the regulation of neuronal alpha-tubulin production.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by a dominant mutation in the X-linked methyl CpG binding protein 2 (MeCP2) gene. Neuroanatomically, RTT is characterized by a reduction in dendritic arborization and perikaryal size in the brain. MECP2 binds methylated promoters and facilitates assembly of a multiprotein repressor complex that includes Sin3A and the histone deacetylases HDAC1/HDAC2. MeCP2 has recently been found to be downregulated in autistic spectrum disorders such as Angelman syndrome (AS) and RTT, which share some phenotypic manifestations. We have conducted expression analysis of cytoskeleton-related genes in brain tissue of RTT and AS patients. Striking examples of genes with reduced expression were TUBA1B and TUBA3 that encode the ubiquitous alpha-tubulin and the neuronal specific alpha-tubulin, respectively. In accordance with the downregulation of expression of these genes, we have observed a reduction in the level of the corresponding protein product-tyrosinated alpha-tubulin. Low levels of alpha-tubulin and deteriorated cell morphology were also observed in MeCP2(-/y) MEF cells. The effects of MeCP2 deficiency in these cells were completely reversed by introducing and expressing the human MeCP2 gene. These results imply that MeCP2 is involved in the regulation of neuronal alpha-tubulin and add molecular evidence that reversal of the effects of MeCP2 deficiency is achievable. This raises hopes for a cure of Rett syndrome and related MeCP2 deficiency disorders of the autistic spectrum.

MeCP2 deficiency in the brain decreases BDNF levels by REST/CoREST-mediated repression and increases TRKB production.

Disruptions in the expression of the BDNF gene that encodes a neurotrophic factor involved in neuronal survival, differentiation and synaptic plasticity has been proposed to contribute to the molecular pathogenesis of Rett syndrome. Rett syndrome (RTT) is a neurodevelopmental disorder, caused by mutations in the X-linked methyl CpG binding protein 2 gene (MeCP2). MeCP2 deficiency in the brain has been shown to decrease overall expression of BDNF in spite of an observed increase in the activity of promoter III that appears to be controlled directly by MeCP2. Therefore, how MeCP2 deficiency causes an overall downregulation of BDNF expression was an enigma. Here we report that MeCP2 deficiency in human and mouse brain causes an increase in expression of two neuronal gene transcriptional repressors REST (RE1 silencing transcription factor), and CoREST. MeCP2 binds to and is involved in repression of Rest and CoRest promoters despite their unmethylated state. MeCP2 depletion is associated with a change in the histone modification profile to a more active conformation. The elevated levels of REST and CoREST in the brain of RTT patients and MeCP2 deficient mice result in downregulation of BDNF, apparently by their binding to the RE1 (element) located between the first two promoters of the BDNF gene. Interestingly, the NTRK2 gene that encodes the BDNF receptor, TRKB, was overexpressed in MeCP2 deficient human and mouse brains either directly or as an attempt to compensate for BDNF deficiency.

IKAP/hELP1 deficiency in the cerebrum of familial dysautonomia patients results in down regulation of genes involved in oligodendrocyte differentiation and in myelination.

The gene affected in the congenital neuropathy familial dysautonomia (FD) is IKBKAP that codes for the IKAP/hELP1 protein. Several different functions have been suggested for this protein, but none of them have been verified in vivo or shown to have some link with the FD phenotype. In an attempt to elucidate the involvement of IKAP/hELP1 in brain function, we searched for IKAP/hELP1 target genes associated with neuronal function. In a microarray expression analysis using RNA extracted from the cerebrum of two FD patients as well as sex and age matched controls, no genes were found to be upregulated in the FD cerebrum. However, 25 genes were downregulated more than 2-fold in the cerebrum of both the male FD child and female FD mature woman. Thirteen of them are known to be involved in oligodendrocyte development and myelin formation. The down regulation of all these genes was verified by real-time PCR. Four of these genes were also confirmed to be downregulated at the protein level. These results are statistically significant and have high biological relevance, since seven of the downregulated genes in the cerebrum of the FD patients were shown by others to be upregulated during oligodendrocyte differentiation in vitro. Our results therefore suggest that IKAP/hELP1 may play a role in oligodendrocyte differentiation and/or myelin formation.

Splicing mutation associated with Rett syndrome and an experimental approach for genetic diagnosis.

Around 80% of Rett syndrome (RS) cases have a mutation or deletion within the coding sequence of the MeCP2 gene. The other RS patients remain genetically undiagnosed. A significant fraction (10-15%) of disease-causing mutations in humans, affect pre-mRNA splicing. Two potential splice mutations were found in the MeCP2 gene in RS patients, however it was not clear whether these mutations in fact interfere with splicing and consequently cause RS. One such mutation is a deletion of the GT dinucleotide at the 5' donor splice site of exon 1 and the other a deletion of the T nucleotide in the polypyrimidine tract (PPT) of intron 3. Here we experimentally assess the effects exerted by these mutations on the expression of MeCP2 in patients' blood samples and on splicing of the MeCP2 transcript using a hybrid minigene in transient transfection experiments. The results revealed that the Delta T mutation in the PPT is a benign polymorphism and that the GT deletion in intron 1 is a bona fide splicing mutation that causes a complete skipping of exon 1 in the minigene transfection experiment. This explains the observed complete elimination of the MeCP2 message and protein in the lymphoblast clones of the RS patient that carry the mutation on the active X. An analysis of the MeCP2 transcript and protein production in lymphoblast clones, as described here, can be used to confirm clinically diagnosed RS patients with no mutation in the MeCP2 coding sequence. This will enable RS diagnosis without specifically identifying a mutation.

DNA methylation in epigenetic control of gene expression.

Over three decades ago DNA methylation had been suggested to play a role in the regulation of gene expression. This chapter reviews the development of this field of research over the last three decades, from the time when this idea was proposed up until now when the molecular mechanisms involved in the effect of DNA methylation on gene expression are becoming common knowledge. The dynamic changes that the DNA methylation pattern undergoes during gametogenesis and embryo development have now been revealed. The three-way connection between DNA methylation, chromatin structure and gene expression has been recently clarified and the interrelationships between DNA methylation and histone modification are currently under investigation. DNA methylation is implicated in developmental processes such as X-chromosome inactivation, genomic imprinting and disease, including tumor development. This chapter discusses all these issues in depth.

MeCP2 deficiency in Rett syndrome causes epigenetic aberrations at the PWS/AS imprinting center that affects UBE3A expression.

Rett syndrome (RS) is a severe and progressive neurodevelopmental disorder caused by heterozygous mutations in the X-linked methyl CpG binding protein 2 (MeCP2) gene. MeCP2 is a nuclear protein that binds specifically to methylated DNA and functions as a general transcription repressor in the context of chromatin remodeling complexes. RS shares clinical features with those of Angelman syndrome (AS), an imprinting neurodevelopmental disorder. In AS patients, the maternally expressed copy of UBE3A that codes for the ubiquitin protein ligase 3A (E6-AP) is repressed. The similar phenotype of these two syndromes led us to hypothesize that part of the RS phenotype is due to MeCP2-associated silencing of UBE3A. Indeed, UBE3A mRNA and protein are shown here to be significantly reduced in human and mouse MECP2 deficient brains. This reduced UBE3A level was associated with biallelic production of the UBE3A antisense RNA. In addition, MeCP2 deficiency resulted in elevated histone H3 acetylation and H3(K4) methylation and reduced H3(K9) methylation at the PWS/AS imprinting center, with no effect on DNA methylation or SNRPN expression. We conclude, therefore, that MeCP2 deficiency causes epigenetic aberrations at the PWS imprinting center. These changes in histone modifications result in loss of imprinting of the UBE3A antisense gene in the brain, increase in UBE3A antisense RNA level and, consequently reduction in UBE3A production.

Establishing the epigenetic status of the Prader-Willi/Angelman imprinting center in the gametes and embryo.

The Prader-Willi/Angelman imprinted domain on human chromosome 15q11-q13 is regulated by an imprinting control center (IC) composed of a sequence around the SNRPN promoter (PWS-SRO) and a sequence located 35 kb upstream (AS-SRO). We have previously hypothesized that the primary imprint is established on AS-SRO, which then confers imprinting on PWS-SRO. Here we examine this hypothesis using a transgene that includes both AS-SRO and PWS-SRO sequences and carries out the entire imprinting process. The epigenetic features of this transgene resemble those previously observed on the endogenous locus, thus allowing analyses in the gametes and early embryo. We demonstrate that the primary imprint is in fact established in the gametes, creating a differentially methylated CpG cluster (DMR) on AS-SRO, presumably by an adjacent de novo signal (DNS). The DMR and DNS bind specific proteins: an allele-discrimination protein (ADP) and a de novo methylation protein, respectively. ADP, being a maternal protein, is involved in both the establishment of DMR in the gametes and in its maintenance through implantation when methylation of PWS-SRO on the maternal allele takes place. Importantly, while the AS-SRO is required in the gametes to confer methylation on PWS-SRO, it is dispensable later in development.

Control elements within the PWS/AS imprinting box and their function in the imprinting process.

A cluster of imprinted genes on human chromosome 15q11-q13 (the PWS/AS domain) and its ortholog on mouse chromosome 7c is believed to be regulated by an imprinting control center. Although minideletions in this region in Angelman syndrome (AS) and Prader-Willi syndrome (PWS) patients revealed that two elements, shortest deletion regions of overlap in AS families and PWS families (AS-SRO and PWS-SRO), respectively, constitute the IC, the molecular mechanism that governs this regional control remains obscure. To understand how this imprinting center works, a mouse model was sought. The striking similarity between the human and mouse sequences allowed the generation of a minitransgene (AS-SMP) composed of AS-SRO and the Snrpn minimal promoter (SMP) the mouse ortholog of PWS-SRO. This minitransgene carries out, in a highly reliable and reproducible manner, all steps of the imprinting process. In an attempt to decipher the molecular mechanism of the imprinting process, we generated and tested for imprinting five minitransgenes based on AS-SMP, in which various parts of the 160 bp SMP were deleted. These experiments revealed a set of five cis elements that carry out the various steps of the imprinting process. This set includes: (i). two copies of a de novo methylation signal (DNS) that establish the maternal imprint during oogenesis; (ii). an allele discrimination signal that establishes the paternal imprint; and (iii). two elements that act together to maintain the paternal imprint. Two functionally redundant sets of the five elements were found on the respective endogenous mouse sequence explaining the previously published contradictory results of targeted deletion experiments. Together with the fact that all five elements bind specific proteins that are presumably the factors acting in trans in the imprinting process, our observations set the stage for a comprehensive study of the molecular mechanism involved in the control of the imprinting process.

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo.

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.

Characterization of the human Snrpn minimal promoter and cis elements within it.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by loss of gene function of the imprinted genes including Snrpn within a 2 Mb domain on chromosome 15q11-13. Based on microdeletions in PWS and AS patients, a 4.3 sequence around Snrpn promoter/exon 1, together with a 880 bp sequence upstream to Snrpn, are believed to encompass an imprinting control center for the entire 2 Mb domain. We have previously characterized the mouse Snrpn minimal promoter and a 7 bp element (SBE) within it, which is required for its activity. Here we describe the human Snrpn minimal promoter sequence, which is comprised of a 71 bp upstream sequence and 51 bp of exon 1. The SBE, which has been shown to be critical for mouse promoter activity, is also found in the human sequence and absolutely required for promoter activity. Methylation of this element, like in the mouse, prevents the binding of a protein factor and abolishes promoter activity. In addition, the 5' end of exon 1 must contain cis elements that support promoter activity. In contrast, the 3' end of exon 1 appears to repress promoter activity. This sequence specifically binds a protein factor which presumably exerts a repressory effect on the promoter. Methylation of this sequence prevents the binding of this protein.