Patricia Cavelier

Chromatin Remodeling at the Ig Loci Prior to V(D)J Recombination

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and κ L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain JH cluster and increased DNase I sensitivity of VH and JH segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of VH segments and a reciprocal increase in the nuclease sensitivity of Vκ and Jκ segments. In contrast, JH segments remain DNase I sensitive, and their nucleosomal organization is maintained in μ+ recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and κ L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.

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 VHH and CHH, respectively, and which differ from conventional VH and CH 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 VHH and VH genes followed by a unique DH-JH cluster and C region genes, which include both CHH and CH genes. Although this general gene organization greatly resembles that of other typical mammalian Vn-Dn-Jn-Cn 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.

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 VH 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.

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.

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.

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.

Comparison of Mouse and Rabbit Eiκ Enhancers Indicates That Different Elements Within the Enhancer May Mediate Activation of Transcription and Recombination

The intronic Ig κ-light chain enhancer (Eiκ) has been implicated in regulation of transcription and Vκ-Jκ recombination at the κ locus. To identify sequences within the Eiκ enhancer which are involved in control of recombination, we have made use of the finding that the Eiκ element from the rabbit b9 κ locus is capable of inducing rearrangement, but not transcription of κ genes in mouse lymphoid cells. We have therefore compared the binding of murine nuclear proteins to the mouse and rabbit Eiκ elements. DNase I footprinting and gel mobility shift assays indicate that only the κB, κE1, and κE2 sites of the rabbit enhancer are able to interact with murine trans-acting factors. Moreover, although the rabbit κB site binds murine NF-κB p50/p50 and p50/p65 complexes with high affinity, this site is not capable of mediating transcriptional activation of transient transfection reporter constructs in mouse B lineage cells. These results therefore suggest that, in contrast to the maintenance of κ enhancer transcription which requires all of the Eiκ sites, only the κB, κE1, and κE2 sites may be necessary for the recombinational activity of the enhancer. Furthermore, NF-κB-mediated effects on transcription and recombination appear to involve separate downstream activation pathways.

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 (&bgr; = –0.406, P = .001, R2 = 0.206) and length increase (&bgr; = –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 (&bgr; = 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 (&bgr; = –0.331, P = .002, R2 = 0.165; and &bgr; = –0.307, P = .005, R2 = 0.159, respectively). RTL1 expression, in addition, was negatively linked to birth length (&bgr; = –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.

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

Abstract 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

ABSTRACT 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. Summary: Alkaline phosphatase activity and immunochemistry reveal that oyster germline cells are located in the gonad duct outer edge during the resting period and proliferate very early during the next sexual cycle.