EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos.

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2(-/-) embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development.

Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation.

BACKGROUND: Upon maturation in the bone marrow, polyploid megakaryocytes elongate very long and thin cytoplasmic branches called proplatelets. Proplatelets enter the sinusoids blood vessels in which platelets are ultimately released. Microtubule dynamics, bundling, sliding, and coiling, drive these dramatic morphological changes whose regulation remains poorly understood. Microtubule properties are defined by tubulin isotype composition and post-translational modification patterns. It remains unknown whether microtubule post-translational modifications occur in proplatelets and if so, whether they contribute to platelet formation. RESULTS: Here, we show that in proplatelets from mouse megakaryocytes, microtubules are both acetylated and polyglutamylated. To bypass the difficulties of working with differentiating megakaryocytes, we used a cell model that allowed us to test the functions of these modifications. First, we show that α2bß3integrin signaling in D723H cells is sufficient to induce ß1tubulin expression and recapitulate the specific microtubule behaviors observed during proplatelet elongation and platelet release. Using this model, we found that microtubule acetylation and polyglutamylation occur with different spatio-temporal patterns. We demonstrate that microtubule acetylation, polyglutamylation, and ß1tubulin expression are mandatory for proplatelet-like elongation, swelling formation, and cytoplast severing. We discuss the functional importance of polyglutamylation of ß1tubulin-containing microtubules for their efficient bundling and coiling during platelet formation. CONCLUSIONS: We characterized and validated a powerful cell model to address microtubule behavior in mature megakaryocytes, which allowed us to demonstrate the functional importance of microtubule acetylation and polyglutamylation for platelet release. Furthermore, we bring evidence of a link between the expression of a specific tubulin isotype, the occurrence of microtubule post-translational modifications, and the acquisition of specific microtubule behaviors. Thus, our findings could widen the current view of the regulation of microtubule behavior in cells such as osteoclasts, spermatozoa, and neurons, which express distinct tubulin isotypes and display specific microtubule activities during differentiation.

In Vitro Corticogenesis from Embryonic Stem Cells Recapitulates the In Vivo Epigenetic Control of Imprinted Gene Expression.

In vitro corticogenesis from embryonic stem cells (ESCs) is an attractive model of cortical development and a promising tool for cortical therapy. It is unknown to which extent epigenetic mechanisms crucial for cortex development and function, such as parental genomic imprinting, are recapitulated by in vitro corticogenesis. Here, using genome-wide transcriptomic and methylation analyses on hybrid mouse tissues and cells, we find a high concordance of imprinting status between in vivo and ESC-derived cortices. Notably, in vitro corticogenesis strictly reproduced the in vivo parent-of-origin-dependent expression of 41 imprinted genes (IGs), including Mest and Cdkn1c known to control corticogenesis. Parent-of-origin-dependent DNA methylation was also conserved at 14 of 18 imprinted differentially methylated regions. The least concordant imprinted locus was Gpr1-Zdbf2, where the aberrant bi-allelic expression of Zdbf2 and Adam23 was concomitant with a gain of methylation on the maternal allele in vitro. Combined, our data argue for a broad conservation of the epigenetic mechanisms at imprinted loci in cortical cells derived from ESCs. We propose that in vitro corticogenesis helps to define the still poorly understood mechanisms that regulate imprinting in the brain and the roles of IGs in cortical development.

Early gametogenesis in the Pacific oyster: new insights using stem cell and mitotic markers.

While our knowledge of bivalve gametogenesis has progressed in recent times, more molecular markers are needed in order to develop tissue imaging. Here, we identified stem cell and mitotic markers to further characterize oyster early gametogenesis, mainly through immunofluorescence microscopy. Intense alkaline phosphatase activity, a non-specific marker for stem cells, was detected on the outer edge of the gonad ducts at the post-spawning stage, suggesting an abundance of undifferentiated cells very early during the sexual cycle. This observation was confirmed using an antibody against Sox2, a transcription factor specific for stem or germline cells, which labeled cells in the gonad duct inner mass and ciliated epithelium early during the initial oyster sexual cycle. Moreover, Vasa, a cytoplasmic marker for germline cells, was also detected in the gonad acini and duct cells, thus confirming that germline cells were abundant early on. In addition, the binding of the minichromosome maintenance MCM6 protein to chromatin indicated the gonad acini and duct cells were engaged in the cell cycle. DNA replication was indeed confirmed by an abundant in vivo incorporation of BrdU into the duct cell chromatin. Finally, proliferation of acini and duct cells was demonstrated by the chromatin-bound Ser10-phosphorylated histone H3, a mitotic marker. The markers for the cell cycle and mitosis used here thus indicate that acini and duct cells were already actively dividing early during the oyster sexual cycle. In addition, together with the stem cell markers, these data reveal that the epithelium delimiting the duct outer edge contains a dynamic population of undifferentiated cells.

The placental imprinted DLK1-DIO3 domain: a new link to prenatal and postnatal growth in humans.

BACKGROUND: The developmentally important DLK1-DIO3 imprinted domain on human chromosome 14 is regulated by 2 differentially methylated regions, the intergenic differentially methylated region and the MEG3 differentially methylated region. OBJECTIVE: The aim was to determine the natural variation in DNA methylation at these differentially methylated regions in human placentas, and to determine its link to gene expression levels at the domain. The second goal was to explore whether the domain's methylation and gene expression correlate with prenatal and early postnatal growth of the conceptus. STUDY DESIGN: Using pyrosequencing, we determined methylation levels at CpG dinucleotides across the 2 regulatory differentially methylated regions in placentas from 91 healthy mothers. At birth, placentas and infants were weighed (gestational age 39 ± 1 weeks; birthweight SD score 0.1 ± 0.8) and placental biopsies were collected. RNA expression was quantitated by real-time polymerase chain reaction. Infants' weights and lengths were followed up monthly during the first year. RESULTS: Methylation levels at the 2 regulatory differentially methylated regions were linked and varied considerably between placentas. MEG3 promoter differentially methylated region methylation correlated negatively with weight increase (ß = -0.406, P = .001, R2 = 0.206) and length increase (ß = -0.363, P = .002, R2 = 0.230) during the first postnatal year. The methylation level of the intergenic differentially methylated region correlated with DIO3 expression (ß = 0.313, P = .032, R2 = 0.152). Furthermore, the expression of both DIO3 and RTL1 (both imprinted genes within the DLK1-DIO3 domain) was negatively associated with birthweight (ß = -0.331, P = .002, R2 = 0.165; and ß = -0.307, P = .005, R2 = 0.159, respectively). RTL1 expression, in addition, was negatively linked to birth length (ß = -0.306, P = .007, R2 = 0.162). CONCLUSION: Our combined findings strongly suggest that placental DNA methylation at the DLK1-DIO3 domain's intergenic differentially methylated region and MEG3 promoter differentially methylated region relates to measures of early human growth, and may thus contribute to its control.

Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.

An in vitro model to assess the immunosuppressive effect of tick saliva on the mobilization of inflammatory monocyte-derived cells.

Tick-borne pathogens cause potent infections. These pathogens benefit from molecules contained in tick saliva that have evolved to modulate host innate and adaptive immune responses. This is called "saliva-activated transmission" and enables tick-borne pathogens to evade host immune responses. Ticks feed on their host for relatively long periods; thus, mechanisms counteracting the inflammation-driven recruitment and activation of innate effector cells at the bite site, are an effective strategy to escape the immune response. Here, we developed an original in vitro model to evaluate and to characterize the immunomodulatory effects of tick saliva that prevent the establishment of a local inflammatory immune response. This model mimics the tick bite and enables the assessment of the effect of saliva on the inflammatory-associated dynamic recruitment of cells from the mononuclear phagocyte system. Using this model, we were able to recapitulate the dual effect of tick saliva on the mobilization of inflammatory monocyte-derived cells, i.e. (i) impaired recruitment of monocytes from the blood to the bite wound; and (ii) poor mobilization of monocyte-derived cells from the skin to the draining lymph node. This simple tool reconstitutes the effect of tick saliva in vivo, which we characterized in the mouse, and should enable the identification of important factors facilitating pathogen infection. Furthermore, this model may be applied to the characterization of any pathogen-derived immunosuppressive molecule affecting the establishment of the inflammatory immune response.

Adult somatic progenitor cells and hematopoiesis in oysters.

Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills.

Tetrameric and homodimeric camelid IgGs originate from the same IgH locus.

In addition to producing conventional tetrameric IgGs, camelids have the particularity of producing a functional homodimeric IgG type lacking L (light) chains and only made up of two H (heavy) chains. This nonconventional IgG type is characterized by variable and constant regions referred to as V(H)H and C(H)H, respectively, and which differ from conventional V(H) and C(H) counterparts. Although the structural properties of homodimeric IgGs have been well investigated, the genetic bases involved in their generation are still largely unknown. In this study, we characterized the organization of genes coding for the H chains of tetrameric and homodimeric IgGs by constructing an alpaca (Lama pacos) genomic cosmid library. We showed that a single IgH locus in alpaca chromosome 4 contains all of the genetic elements required for the generation of the two types of Igs. The alpaca IgH locus is composed of a V region that contains both V(H)H and V(H) genes followed by a unique D(H)-J(H) cluster and C region genes, which include both C(H)H and C(H) genes. Although this general gene organization greatly resembles that of other typical mammalian V(n)-D(n)-J(n)-C(n) translocon IgH loci, the intermixed gene organization within the alpaca V and C regions reveals a new type of translocon IgH locus. Furthermore, analyses of cDNA coding for the membrane forms of IgG and IgM present in alpaca peripheral blood B cells are most consistent with the notion that the development of a B cell bearing homodimeric IgG passes through an IgM(+) stage, similar to the case for conventional IgG.

Lamin C Counteracts Glucose Intolerance in Aging, Obesity, and Diabetes Through ß-Cell Adaptation.

Aging-dependent changes in tissue function are associated with the development of metabolic diseases. However, the molecular connections linking aging, obesity, and diabetes remain unclear. Lamin A, lamin C, and progerin, products of the Lmna gene, have antagonistic functions on energy metabolism and life span. Lamin C, albeit promoting obesity, increases life span, suggesting that this isoform is crucial for maintaining healthy conditions under metabolic stresses. Because ß-cell loss during obesity or aging leads to diabetes, we investigated the contribution of lamin C to ß-cell function in physiopathological conditions. We demonstrate that aged lamin C only-expressing mice (Lmna LCS/LCS ) become obese but remain glucose tolerant due to adaptive mechanisms including increased ß-cell mass and insulin secretion. Triggering diabetes in young mice revealed that Lmna LCS/LCS animals normalize their fasting glycemia by both increasing insulin secretion and regenerating ß-cells. Genome-wide analyses combined to functional analyses revealed an increase of mitochondrial biogenesis and global translational rate in Lmna LCS/LCS islets, two major processes involved in insulin secretion. Altogether, our results demonstrate for the first time that the sole expression of lamin C protects from glucose intolerance through a ß-cell-adaptive transcriptional program during metabolic stresses, highlighting Lmna gene processing as a new therapeutic target for diabetes treatment.

A novel mouse c-fos intronic promoter that responds to CREB and AP-1 is developmentally regulated in vivo.

BACKGROUND: The c-fos proto-oncogene is an archetype for rapid and integrative transcriptional activation. Innumerable studies have focused on the canonical promoter, located upstream from the transcriptional start site. However, several regulatory sequences have been found in the first intron. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely conserved region in c-fos first intron that contains a putative TATA box, and functional TRE and CRE sites. This fragment drives reporter gene activation in fibroblasts, which is enhanced by increasing intracellular calcium and cAMP and by cotransfection of CREB or c-Fos/c-Jun expression vectors. We produced transgenic mice expressing a lacZ reporter controlled by the intronic promoter. Lac Z expression of this promoter is restricted to the developing central nervous system (CNS) and the mesenchyme of developing mammary buds in embryos 12.5 days post-conception, and to brain tissue in adults. RT-QPCR analysis of tissue mRNA, including the anlage of the mammary gland and the CNS, confirms the existence of a novel, nested mRNA initiated in the first intron. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence for a novel, developmentally regulated promoter in the first intron of the c-fos gene.

Activation of V(D)J recombination at the IgH chain JH locus occurs within a 6-kilobase chromatin domain and is associated with nucleosomal remodeling.

IgH genes are assembled during early B cell development by a series of regulated DNA recombination reactions in which DH and JH segments are first joined followed by V(H) to DJH rearrangement. Recent studies have highlighted the role of chromatin structure in the control of V(D)J recombination. In this study, we show that, in murine pro-B cell precursors, the JH segments are located within a 6-kb DNase I-sensitive chromatin domain containing acetylated histones H3 and H4, which is delimited 5' by the DQ52 promoter element and 3' by the intronic enhancer. Within this domain, the JH segments are covered by phased nucleosomes. High-resolution mapping of nucleosomes reveals that, in pro-B cells, unlike recombination refractory nonlymphoid cells, the recombination signal sequences flanking the four JH segments are located in regions of enhanced micrococcal nuclease and restriction enzyme accessibility, corresponding to either nucleosome-free regions or DNA rendered accessible within a nucleosome. These results support the idea that nucleosome remodeling provides an additional level of control in the regulation of Ig locus accessibility to recombination factors in B cell precursors.