Isabelle Robert

PARP-3 and APLF Function Together to Accelerate Nonhomologous End-Joining

PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.

Poly(ADP-ribose) polymerases in double-strand break repair: focus on PARP1, PARP2 and PARP3.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.

Epigenetic tethering of AID to the donor switch region during immunoglobulin class switch recombination.

A complex of KAP1 and HP1 is needed to tether AID to the H3K9me3-marked donor switch region during CSR.

The IgH 3′ regulatory region controls somatic hypermutation in germinal center B cells

Somatic hypermutation in variable heavy chain rearranged regions is abrogated in the absence of the 3′ regulatory region enhancer, whereas transcription rate in the Ig heavy chain is only partially reduced.

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56(+) cells grew rapidly, a population of CD15(+) cells emerged, partly from CD56(+) cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56(+) and CD15(+) cells shared osteogenic and chondrogenic abilities, while CD56(+) cells presented a myogenic capacity and CD15(+) cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

Functional aspects of PARylation in induced and programmed DNA repair processes: preserving genome integrity and modulating physiological events.

To cope with the devastating insults constantly inflicted to their genome by intrinsic and extrinsic DNA damaging sources, cells have evolved a sophisticated network of interconnected DNA caretaking mechanisms that will detect, signal and repair the lesions. Among the underlying molecular mechanisms that regulate these events, PARylation catalyzed by Poly(ADP-ribose) polymerases (PARPs), appears as one of the earliest post-translational modification at the site of the lesion that is known to elicit recruitment and regulation of many DNA damage response proteins. In this review we discuss how the complex PAR molecule operates in stress-induced DNA damage signaling and genome maintenance but also in various physiological settings initiated by developmentally programmed DNA breakage. To illustrate the latter, particular emphasis will be placed on the emerging contribution of PARPs to B cell receptor assembly and diversification.

The cohesin complex regulates immunoglobulin class switch recombination

The cohesin complex is recruited to DNA switch regions and is required for nonhomologous end joining during immunoglobulin class switch recombination.

Prospective flow cytometric evaluation of nucleated red blood cells in cord blood units and relationship with nucleated and CD34+ cell quantification

BACKGROUND:  Cord blood (CB) represents an alternate source of stem cells in transplantation. Nucleated red blood cells (NRBCs) are a physiological subset of CB population. Although it is important to have an accurate estimate of CD34+ cell number, NRBCs could compromise white blood cell count and interfere with CD34+ cell quantification.

A novel untranslated 'exon H' of the human choline acetyltransferase gene in placenta.

To investigate the existence of 5′‐region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5′‐untranslated regions (UTR) in human neuronal and non‐neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG‐3 and neuroblastoma CHP126 and MC‐IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT‐PCR). We used a sense primer located in the 5′‐flanking region, in the previously defined intronic sequence and an anti‐sense primer located in the common coding exon 2 of the hChAT gene. An amplified product of 567 bp in size was obtained only in human placenta and in JEG‐3 cells whereas it was absent in spinal cord, CHP126 and MC‐IXC cells. It was designated ‘H‐type’ of ChAT mRNA. Whereas CHP126 produced the R‐ and N‐type of ChAT mRNAs, no transcript of the N‐and R‐type was detected in JEG‐3 and human placenta. In addition, CHP126 and JEG‐3 cells and placenta showed the expression of the M‐type of ChAT mRNA. The idendity of the amplified 567 bp product (H‐type) was confirmed by Southern hybridization and sequencing. The nucleotide sequence of the amplified fragment in placenta revealed the existence of a previously unknown type of ChAT mRNA produced by alternative splicing. Using primer extension we further determined the transcription initiation site of the H‐type hChAT mRNA in placenta. These results demonstrate the expression of a novel ChAT mRNA isoform in human placenta in addition to the M‐type. These data may be possibly explained by the presence of a placenta specific promoter in the ChAT gene, which might be the proximal promoter P1.

Synergistic activation of the human choline acetyltransferase gene by c-Myb and C/EBPβ

To elucidate regulatory mechanisms at the transcriptional level of the human choline acetyltransferase gene (hChAT) we performed cotransfections assays in NG108-15 and SN56 cells using ChAT-CAT reporter plasmids with c-Myb and C/EBPbeta expression plasmids. The hChAT gene has several promoters, one of which (promoter P2 or M-type) is both c-Myb and C/EBPbeta inducible as 3-4-fold trans-activation was obtained in both cell lines when using either c-Myb or C/EBPbeta expression vectors alone. The simultaneous expression of c-Myb and C/EBPbeta in the absence or presence of NGFI-C (egr4) leads respectively to a 15-fold and 32-fold synergistic transcriptional activation of promoter P2. In the region upstream of exon M (P2) we identified a functional composite element including a c-Myb next to a C/EBP binding site. An oligonucleotide containing the composite element confers c-Myb and C/EBPbeta responsiveness to a heterologous promoter which is reduced after mutation of the c-Myb binding site. We also show that the coactivators CBP/p300 are required for c-Myb and C/EBPbeta trans-activation function and that RARalpha, RXRalpha and T3R have an inhibitory action on the synergistic transcriptional activity of c-Myb and C/EBPbeta and propose a model to explain the phenomena. Taken together, the results suggest that the synergistic effect of c-Myb and C/EBPbeta, previously observed in the hematopoietic system, functions equally in the neuronal system.