ASC-1 Is a Cell Cycle Regulator Associated with Severe and Mild Forms of Myopathy.

OBJECTIVE: Recently, the ASC-1 complex has been identified as a mechanistic link between amyotrophic lateral sclerosis and spinal muscular atrophy (SMA), and 3 mutations of the ASC-1 gene TRIP4 have been associated with SMA or congenital myopathy. Our goal was to define ASC-1 neuromuscular function and the phenotypical spectrum associated with TRIP4 mutations. METHODS: Clinical, molecular, histological, and magnetic resonance imaging studies were made in 5 families with 7 novel TRIP4 mutations. Fluorescence activated cell sorting and Western blot were performed in patient-derived fibroblasts and muscles and in Trip4 knocked-down C2C12 cells. RESULTS: All mutations caused ASC-1 protein depletion. The clinical phenotype was purely myopathic, ranging from lethal neonatal to mild ambulatory adult patients. It included early onset axial and proximal weakness, scoliosis, rigid spine, dysmorphic facies, cutaneous involvement, respiratory failure, and in the older cases, dilated cardiomyopathy. Muscle biopsies showed multiminicores, nemaline rods, cytoplasmic bodies, caps, central nuclei, rimmed fibers, and/or mild endomysial fibrosis. ASC-1 depletion in C2C12 and in patient-derived fibroblasts and muscles caused accelerated proliferation, altered expression of cell cycle proteins, and/or shortening of the G0/G1 cell cycle phase leading to cell size reduction. INTERPRETATION: Our results expand the phenotypical and molecular spectrum of TRIP4-associated disease to include mild adult forms with or without cardiomyopathy, associate ASC-1 depletion with isolated primary muscle involvement, and establish TRIP4 as a causative gene for several congenital muscle diseases, including nemaline, core, centronuclear, and cytoplasmic-body myopathies. They also identify ASC-1 as a novel cell cycle regulator with a key role in cell proliferation, and underline transcriptional coregulation defects as a novel pathophysiological mechanism. ANN NEUROL 2020;87:217-232.

Inherited Defects of the ASC-1 Complex in Congenital Neuromuscular Diseases.

Defects in transcriptional and cell cycle regulation have emerged as novel pathophysiological mechanisms in congenital neuromuscular disease with the recent identification of mutations in the TRIP4 and ASCC1 genes, encoding, respectively, ASC-1 and ASCC1, two subunits of the ASC-1 (Activating Signal Cointegrator-1) complex. This complex is a poorly known transcriptional coregulator involved in transcriptional, post-transcriptional or translational activities. Inherited defects in components of the ASC-1 complex have been associated with several autosomal recessive phenotypes, including severe and mild forms of striated muscle disease (congenital myopathy with or without myocardial involvement), but also cases diagnosed of motor neuron disease (spinal muscular atrophy). Additionally, antenatal bone fractures were present in the reported patients with ASCC1 mutations. Functional studies revealed that the ASC-1 subunit is a novel regulator of cell cycle, proliferation and growth in muscle and non-muscular cells. In this review, we summarize and discuss the available data on the clinical and histopathological phenotypes associated with inherited defects of the ASC-1 complex proteins, the known genotype-phenotype correlations, the ASC-1 pathophysiological role, the puzzling question of motoneuron versus primary muscle involvement and potential future research avenues, illustrating the study of rare monogenic disorders as an interesting model paradigm to understand major physiological processes.

The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease.

Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation in TRIP4 encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted in TRIP4 mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and in Trip4 knocked-down C2C12 led to a significant reduction in myotube diameter ex vivo and in vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the first TRIP4 mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism.

The p.R482W substitution in A-type lamins deregulates SREBP1 activity in Dunnigan-type familial partial lipodystrophy.

Nuclear lamins are involved in many cellular functions due to their ability to bind numerous partners including chromatin and transcription factors, and affect their properties. Dunnigan type familial partial lipodystrophy (FPLD2; OMIM#151660) is caused in most cases by the A-type lamin R482W mutation. We report here that the R482W mutation affects the regulatory activity of sterol response element binding protein 1 (SREBP1), a transcription factor that regulates hundreds of genes involved in lipid metabolism and adipocyte differentiation. Using in situ proximity ligation assays (PLA), reporter assays and biochemical and transcriptomic approaches, we show that interactions of SREBP1 with lamin A and lamin C occur at the nuclear periphery and in the nucleoplasm. These interactions involve the Ig-fold of A-type lamins and are favored upon SREBP1 binding to its DNA target sequences. We show that SREBP1, LMNA and sterol response DNA elements form ternary complexes in vitro. In addition, overexpression of A-type lamins reduces transcriptional activity of SREBP1. In contrast, both overexpression of LMNA R482W in primary human preadipocytes and endogenous expression of A-type lamins R482W in FPLD2 patient fibroblasts, reduce A-type lamins-SREBP1 in situ interactions and upregulate a large number of SREBP1 target genes. As this LMNA mutant was previously shown to inhibit adipogenic differentiation, we propose that deregulation of SREBP1 by mutated A-type lamins constitutes one underlying mechanism of the physiopathology of FPLD2. Our data suggest that SREBP1 targeting molecules could be considered in a therapeutic context.

Lamin A/C-promoter interactions specify chromatin state-dependent transcription outcomes.

The nuclear lamina is implicated in the organization of the eukaryotic nucleus. Association of nuclear lamins with the genome occurs through large chromatin domains including mostly, but not exclusively, repressed genes. How lamin interactions with regulatory elements modulate gene expression in different cellular contexts is unknown. We show here that in human adipose tissue stem cells, lamin A/C interacts with distinct spatially restricted subpromoter regions, both within and outside peripheral and intra-nuclear lamin-rich domains. These localized interactions are associated with distinct transcriptional outcomes in a manner dependent on local chromatin modifications. Down-regulation of lamin A/C leads to dissociation of lamin A/C from promoters and remodels repressive and permissive histone modifications by enhancing transcriptional permissiveness, but is not sufficient to elicit gene activation. Adipogenic differentiation resets a large number of lamin-genome associations globally and at subpromoter levels and redefines associated transcription outputs. We propose that lamin A/C acts as a modulator of local gene expression outcome through interaction with adjustable sites on promoters, and that these position-dependent transcriptional readouts may be reset upon differentiation.

Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins.

Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (∆607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain.

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. Providing a link between miRNA-mediated regulation of gene expression and inflammation, our results suggest that the formation of miRNAs may be regulated by 5LO in leukocytes and cancer cells expressing this lipoxygenase.

TDRD3, a novel Tudor domain-containing protein, localizes to cytoplasmic stress granules.

Our previous work has demonstrated that the Tudor domain of the 'survival of motor neuron' protein and the Tudor domain-containing protein 3 (TDRD3) are highly similar and that they both have the ability to interact with arginine-methylated polypeptides. TDRD3 has been identified among genes whose overexpression has a strong predictive value for poor prognosis of estrogen receptor-negative breast cancers, although its precise function remains unknown. TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain. We report here that TDRD3 localizes predominantly to the cytoplasm, where it co-sediments with the fragile X mental retardation protein on actively translating polyribosomes. We also demonstrate that TDRD3 accumulates into stress granules (SGs) in response to various cellular stresses. Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process. Pull down experiments identified five novel TDRD3 interacting partners, most of which are potentially methylated RNA-binding proteins. Our findings revealed that two of these proteins, SERPINE1 mRNA-binding protein 1 and DEAD/H box-3 (a gene often deleted in Sertoli-cell-only syndrome), are also novel constituents of cytoplasmic SGs. Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.

The R439C mutation in LMNA causes lamin oligomerization and susceptibility to oxidative stress.

Dunnigan-type familial partial lipodystrophy (FPLD) is a laminopathy characterized by an aberrant fat distribution and a metabolic syndrome for which oxidative stress has recently been suggested as one of the disease-causing mechanisms. In a family affected with FPLD, we identified a heterozygous missense mutation c.1315C>T in the LMNA gene leading to the p.R439C substitution. Cultured patient fibroblasts do not show any prelamin A accumulation and reveal honeycomb-like lamin A/C formations in a significant percentage of nuclei. The mutation affects a region in the C-terminal globular domain of lamins A and C, different from the FPLD-related hot spot. Here, the introduction of an extra cysteine allows for the formation of disulphide-mediated lamin A/C oligomers. This oligomerization affects the interaction properties of the C-terminal domain with DNA as shown by gel retardation assays and causes a DNA-interaction pattern that is distinct from the classical R482W FPLD mutant. Particularly, whereas the R482W mutation decreases the binding efficiency of the C-terminal domain to DNA, the R439C mutation increases it. Electron spin resonance spectroscopy studies show significantly higher levels of reactive oxygen species (ROS) upon induction of oxidative stress in R439C patient fibroblasts compared to healthy controls. This increased sensitivity to oxidative stress seems independent of the oligomerization and enhanced DNA binding typical for R439C, as both the R439C and R482W mutants show a similar and significant increase in ROS upon induction of oxidative stress by H2O2.

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.

Human mismatch repair protein MSH6 contains a PWWP domain that targets double stranded DNA.

The eukaryotic mismatch repair (MMR) protein MSH6 exhibits a core region structurally and functionally similar to bacterial MutS. However, it possesses an additional N-terminal region (NTR), comprising a PCNA binding motif, a large region of unknown function and a nonspecific DNA binding fragment. Yeast NTR was recently described as an extended tether between PCNA and the core of MSH6 . In contrast, we show that human NTR presents a globular PWWP domain in the region of unknown function. We demonstrate that this PWWP domain binds double-stranded DNA, without any preference for mismatches or nicks, whereas its apparent affinity for single-stranded DNA is about 20 times lower. The S144I mutation, which in human MSH6 causes inherited somatic defects in MMR resulting in increased development of hereditary non polyposis colorectal cancer , is located in the DNA binding surface of the PWWP domain. However, it only moderately affects domain stability, and it does not perturb DNA binding in vitro.

Combined written and oral information prior to gastrointestinal endoscopy compared with oral information alone: a randomized trial.

BACKGROUND: Little is known about how to most effectively deliver relevant information to patients scheduled for endoscopy. METHODS: To assess the effects of combined written and oral information, compared with oral information alone on the quality of information before endoscopy and the level of anxiety. We designed a prospective study in two Swiss teaching hospitals which enrolled consecutive patients scheduled for endoscopy over a three-month period. Patients were randomized either to receiving, along with the appointment notice, an explanatory leaflet about the upcoming examination, or to oral information delivered by each patient's doctor. Evaluation of quality of information was rated on scales between 0 (none received) and 5 (excellent). The analysis of outcome variables was performed on the basis of intention to treat-analysis. Multivariate analysis of predictors of information scores was performed by linear regression analysis. RESULTS: Of 718 eligible patients 577 (80%) returned their questionnaire. Patients who received written leaflets (N = 278) rated the quality of information they received higher than those informed verbally (N = 299), for all 8 quality-of-information items. Differences were significant regarding information about the risks of the procedure (3.24 versus 2.26, p < 0.001), how to prepare for the procedure (3.56 versus 3.23, p = 0.036), what to expect after the procedure (2.99 versus 2.59, p < 0.001), and the 8 quality-of-information items (3.35 versus 3.02, p = 0.002). The two groups reported similar levels of anxiety before procedure (p = 0.66), pain during procedure (p = 0.20), tolerability throughout the procedure (p = 0.76), problems after the procedure (p = 0.22), and overall rating of the procedure between poor and excellent (p = 0.82). CONCLUSION: Written information led to more favourable assessments of the quality of information and had no impact on patient anxiety nor on the overall assessment of the endoscopy. Because structured and comprehensive written information is perceived as beneficial by patients, gastroenterologists should clearly explain to their patients the risks, benefits and alternatives of endoscopic procedures. TRIAL REGISTRATION: Current Controlled trial number: ISRCTN34382782.

Lamin ChIP from Chromatin Prepared by Micrococcal Nuclease Digestion.

It is now clearly demonstrated that nuclear lamins interact with the genomic DNA and largely contribute to its three-dimensional organization and transcriptional regulation. Emergence of genome-wide mapping techniques such as DamID technology or chromatin immunoprecipitation (ChIP) followed by array hybridization or high-throughput sequencing has allowed the mapping of large lamin-interacting genomic areas called lamina-associated domains. These cover up to 40 % of the genome and are preferentially located in transcriptionally silent heterochromatin at the nuclear periphery. We recently showed that the use of enzymatic rather than physical fragmentation of chromatin in ChIP experiments uncovers new chromatin compartments with features of euchromatin that interacts with A-type lamins. We describe here a detailed ChIP procedure to covalently cross-link protein-DNA, fragment the chromatin fibers by micrococcal nuclease digestion, and solubilize the lamin network with a short sonication pulse prior to immunoprecipitating the lamin-DNA complexes using specific antibodies. Enriched DNA fragments from the lamin-binding sites are then purified as suitable samples for qPCR analysis or high-throughput sequencing.

Distinct features of lamin A-interacting chromatin domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin.

The nuclear lamina has been shown to interact with the genome through lamina-associated domains (LADs). LADs have been identified by DamID, a proximity labeling assay, and more recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs form megabase-size domains at the nuclear periphery, they are gene-poor and mostly heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely bath sonication or digestion with micrococcal nuclease (MNase), leads to the discovery of common but also distinct sets of lamin-interacting domains, or LiDs. Using ChIP-seq, we show the existence of lamin A/C (LMNA) LiDs with distinct gene contents, histone composition enrichment and relationships to lamin B1-interacting domains. The extent of genome coverage of lamin A/C (LMNA) LiDs in sonicated or MNase-digested chromatin is similar (∼730 megabases); however over half of these domains are uniquely detected in sonicated or MNase-digested chromatin. Sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, while MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. LMNB1 LiDs are gene-poor and show no or little enrichment in these marks. Comparison of published LMNB1 DamID LADs with LMNB1 and LMNA LiDs identified here by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin characteristics, and which are not associated with LMNB1. The differential genomic and epigenetic properties of lamin-interacting domains reflect the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible regions presumably localized in the nuclear interior.

Alternatively spliced protein arginine methyltransferase 1 isoform PRMT1v2 promotes the survival and invasiveness of breast cancer cells.

Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types; however, their precise contribution to the cell transformation process is not well understood. We previously reported that the PRMT1 gene generates several alternatively spliced isoforms, and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and subcellular localization. We focus here on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform, and we have found that its relative expression is increased in breast cancer cell lines and tumors. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in an aggressive cancer cell line significantly decreased cell invasion. We also demonstrate that PRMT1v2 overexpression in a non-aggressive cancer cell line was sufficient to render them more invasive. Importantly, this novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. Moreover, this activity requires both proper subcellular localization and methylase activity. Lastly, PRMT1v2 overexpression altered cell morphology and reduced cell-cell adhesion, a phenomenon that we convincingly linked with reduced ß-catenin protein expression. Overall, we demonstrate a specific role for PRMT1v2 in breast cancer cell survival and invasion, underscoring the importance of identifying and characterizing the distinct functional differences between PRMT1 isoforms.

Protein arginine methylation facilitates cotranscriptional recruitment of pre-mRNA splicing factors.

Cotranscriptional recruitment of pre-mRNA splicing factors to their genomic targets facilitates efficient and ordered assembly of a mature messenger ribonucleoprotein particle (mRNP). However, how the cotranscriptional recruitment of splicing factors is regulated remains largely unknown. Here, we demonstrate that protein arginine methylation plays a novel role in regulating this process in Saccharomyces cerevisiae. Our data show that Hmt1, the major type I arginine methyltransferase, methylates Snp1, a U1 small nuclear RNP (snRNP)-specific protein, and that the mammalian Snp1 homolog, U1-70K, is likewise arginine methylated. Genome-wide localization analysis reveals that the deletion of the HMT1 gene deregulates the recruitment of U1 snRNP and its associated components to intron-containing genes (ICGs). In the same context, splicing factors acting downstream of U1 snRNP addition bind to a reduced number of ICGs. Quantitative measurement of the abundance of spliced target transcripts shows that these changes in recruitment result in an increase in the splicing efficiency of developmentally regulated mRNAs. We also show that in the absence of either Hmt1 or of its catalytic activity, an association between Snp1 and the SR-like protein Npl3 is substantially increased. Together, these data support a model whereby arginine methylation modulates dynamic associations between SR-like protein and pre-mRNA splicing factor to promote target specificity in splicing.

Loss of a DNA binding site within the tail of prelamin A contributes to altered heterochromatin anchorage by progerin.

Mutations in the lamin A/C (LMNA) gene that cause Hutchinson-Gilford progeria syndrome (HGPS) lead to expression of a protein called progerin with 50 amino acids deleted from the tail of prelamin A. In cells from patients with HGPS, both the amount and distribution of heterochromatin are altered. We designed in vitro assays to ask whether such alterations might reflect changes in chromatin, DNA and/or histone binding properties of progerin compared to wild-type lamin C-terminal tails. We show that progerin tail has a reduced DNA/chromatin binding capacity and modified trimethylated H3K27 binding pattern, offering a molecular mechanism for heterochromatin alterations related to HGPS.

Alternative splicing yields protein arginine methyltransferase 1 isoforms with distinct activity, substrate specificity, and subcellular localization.

PRMT1 is the predominant member of a family of protein arginine methyltransferases (PRMTs) that have been implicated in various cellular processes, including transcription, RNA processing, and signal transduction. It was previously reported that the human PRMT1 pre-mRNA was alternatively spliced to yield three isoforms with distinct N-terminal sequences. Close inspection of the genomic organization in the 5'-end of the PRMT1 gene revealed that it can produce up to seven protein isoforms, all varying in their N-terminal domain. A detailed biochemical characterization of these variants revealed that unique N-terminal sequences can influence catalytic activity as well as substrate specificity. In addition, our results uncovered the presence of a functional nuclear export sequence in PRMT1v2. Finally, we find that the relative balance of PRMT1 isoforms is altered in breast cancer.

The carboxyl-terminal nucleoplasmic region of MAN1 exhibits a DNA binding winged helix domain.

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.

Hematopoietic stem cells from fancc(-/-) mice have lower growth and differentiation potential in response to growth factors.

Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow (BM) failure. We have previously shown that stem cells from the FA group C mouse model have lower long-term primary and secondary reconstitution ability, and that bone marrow of Fancc(-/-) mice contained fewer lineage-negative (Lin(-))Thy1.2(low)Sca-1(+)c-kit(+) CD34(+) cells but normal levels of Lin(-)Thy1.2(low)Sca-1(+)c-kit(+)CD34(-) primitive cells. These data suggest that CD34(+) primitive cells have either a lower growth or differentiation potential, or that these cells have greater apoptosis levels. To investigate the role Fancc might have on the growth and differentiation potentials of primitive hematopoietic stem cells, we used a single-cell culture system and monitored cell viability, doubling potential, and apoptosis levels of Fancc(-/-) primitive Lin(-)Thy1.2(-)Sca-1(+) (LTS)-CD34(+) and LTS-CD34(-) stem cells. Results showed that Fancc(-/-) LTS-CD34(-) and LTS-CD34(+) cells had altered growth and apoptosis responses to combinations of stimulatory cytokines, most dramatically in response to a combination of factors that included interleukin-3 (IL-3) and IL-6. In addition, Fancc(-/-) LTS-CD34(-) and LTS-CD34(+) cells showed a lower differentiation potential than Fancc(+/+) cells. These results support a role for Fancc in the growth and differentiation of primitive hematopoietic cells and suggest that an altered response to stimulatory cytokines may contribute to BM aplasia in FA patients.