No abstract

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.

Nuclear processes such as transcription, DNA replication, and recombination are dynamically regulated by chromatin structure. Transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent posttranslational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 -an essential component of the RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly 1 -contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease. +To whom correspondence should be addressed: oettinger@frodo.mgh.harvard.edu; ogozani@stanford.edu. * These authors contributed equally to the work Note added in proof: While this work was under review, another study also reported that the RAG2 PHD finger binds to methylated H3K4 30 .Atomic coordinates and structure factors of the RAG2 PHD -H3K4me3 peptide complex have been deposited in the Protein Data Bank with the accession code of 2v89. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Since RAG2 contains a noncanonical plant homeodomain (PHD) finger 6,7 -a module that can mediate interactions with chromatin 8-10 -we asked whether a polypeptide encompassing the RAG2 PHD finger (RAG2 PHD : aa 414-527) can recognize modified histone proteins. In an in vitro screen of peptide microarrays containing ~70 distinct modified histone peptides, we found that RAG2 PHD specifically binds to histone H3 trimethylated at lysine 4 (H3K4me3) ( Fig. 1a ; Fig. S1; data not shown). The specificity of this interaction was confirmed by peptide pulldown assays ( Fig. 1b ; Fig. S2; Fig. S3). RAG2 has a C-terminal extension of 40 aa that is essential for phosphoinositide (PtdInsP)-binding 7 (aa 488-527), but this region is dispensable for H3K4me3-binding as the minimal PHD finger alone (aa 414-487) is sufficient for H3K4me3-recognition (Fig. 1c). In addition, the acidic hinge region of RAG2 (aa 388-412), previously implicated in...

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults. We have analyzed exome sequencing data from 127 individuals with CLL and Sanger sequencing data from 214 additional affected individuals, identifying recurrent somatic mutations in POT1 (encoding protection of telomeres 1) in 3.5% of the cases, with the frequency reaching 9% when only individuals without IGHV@ mutations were considered. POT1 encodes a component of the shelterin complex and is the first member of this telomeric structure found to be mutated in human cancer. Somatic mutation of POT1 primarily occurs in gene regions encoding the two oligonucleotide-/oligosaccharide-binding (OB) folds and affects key residues required to bind telomeric DNA. POT1-mutated CLL cells have numerous telomeric and chromosomal abnormalities that suggest that POT1 mutations favor the acquisition of the malignant features of CLL cells. The identification of POT1 as a new frequently mutated gene in CLL may facilitate novel approaches for the clinical management of this disease.

Hemolytic-uremic syndrome (HUS) is a microvasculature disorder leading to microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of HUS are associated with epidemics of diarrhea caused by verocytotoxin-producing bacteria, but atypical cases of HUS not associated with diarrhea (aHUS) also occur. Early studies describing the association of aHUS with deficiencies of factor H suggested a role for this complement regulator in aHUS. Molecular evidence of factor H involvement in aHUS was first provided by Warwicker et al., who demonstrated that aHUS segregated with the chromosome 1q region containing the factor H gene (HF1) and who identified a mutation in HF1 in a case of familial aHUS with normal levels of factor H. We have performed the mutational screening of the HF1 gene in a novel series of 13 Spanish patients with aHUS who present normal complement profiles and whose plasma levels of factor H are, with one exception, within the normal range. These studies have resulted in the identification of five novel HF1 mutations in four of the patients. Allele HF1 Delta exon2, a genomic deletion of exon 2, produces a null HF1 allele and results in plasma levels of factor H that are 50% of normal. T956M, W1183L, L1189R, and V1197A are missense mutations that alter amino acid residues in the C-terminal portion of factor H, within a region--SCR16-SCR20--that is involved in the binding to solid-phase C3b and to negatively charged cellular structures. This remarkable clustering of mutations in HF1 suggests that a specific dysfunction in the protection of cellular surfaces by factor H is a major pathogenic condition underlying aHUS.

THP1 is a conserved eukaryotic gene whose null mutations confer, in yeast, transcription and genetic instability phenotypes and RNA export defects similar to those of the THO/TREX complex null mutations. In a search for multicopy suppressors of the transcription defect of thp1⌬ cells, we identified the poly(A)؉ RNAbinding heterogeneous nuclear ribonucleoprotein Nab2p. Multicopy NAB2 also suppressed the RNA export defect of thp1⌬ cells. This result suggests a functional relationship between Thp1p and Nab2p. Consistently, the leaky mutation nab2-1 conferred a transcription defect and hyper-recombination phenotype similar to those of thp1⌬, although to a minor degree. Reciprocally, a purified His 6 -tagged Thp1p fusion bound RNA in vitro. In a different approach, we show by Western analyses that a highly purified Thp1p-Sac3p complex does not contain components of THO/TREX and that sac3⌬ confers a transcription defect and hyper-recombination phenotype identical to those of thp1⌬. mRNA degradation was not affected in thp1⌬ mutants, implying that their expression defects are not due to mRNA decay. This indicates that Thp1p-Sac3p is a structural and functional unit. Altogether, our results suggest that Thp1p-Sac3p and Nab2p are functionally related heterogeneous nuclear ribonucleoproteins that define a further link between mRNA metabolism and transcription.In eukaryotes, the processing and export of mRNA from the nuclear transcription site to the cytoplasm require a large number of proteins, a fraction of which act cotranscriptionally. RNA polymerase II (pol II) 1 -driven transcription elongation is coupled with 5Ј-end capping, splicing, 3Ј-end cleavage, and poly(A) ϩ addition (1, 2). Thus, capping enzymes access the 5Ј-end of nascent mRNAs via the pol II C-terminal domain; splicing occurs cotranscriptionally in association with the transcription elongation complex via the pol II C-terminal domain (3-6); and cleavage of the poly(A) ϩ site is dependent on the pol II C-terminal domain (7). The connection between transcription and mRNA metabolism is supported by the accumulation of unspliced pre-mRNAs in mutants with a truncated pol II C-terminal domain (8) or the retention at the site of transcription of human ␤-globin pre-mRNAs defective in either splicing or 3Ј-end formation (9). This connection is also extended to mRNA export. The mRNA is not exported from the nucleus unless the transcript has been properly processed (10).Nuclear mRNA export seems to be connected to transcription at both functional and physical levels. It has been shown that hyperadenylated transcripts accumulate at the site of transcription after different mRNA export blockages (11). Importantly, a conserved TREX complex that contains Sub2p/UAP56, Yra1p/ALY, Tex1p, and the THO complex has been identified in yeast and humans (12). Yra1p/ALY and Sub2p/UAP56 are both involved in mRNA nuclear export (13). THO was previously identified as a four protein-containing complex involved in transcription elongation and the maintenance of genome stability (14). Y...

Although BRCA1 function is essential for maintaining genomic integrity in all cell types, it is unclear why increased risk of cancer in individuals harbouring deleterious mutations in BRCA1 is restricted to only a select few tissues. Here we show that human mammary epithelial cells (HMECs) from BRCA1-mutation carriers (BRCA1mut/+) exhibit increased genomic instability and rapid telomere erosion in the absence of tumour-suppressor loss. Furthermore, we uncover a novel form of haploinsufficiency-induced senescence (HIS) specific to epithelial cells, which is triggered by pRb pathway activation rather than p53 induction. HIS and telomere erosion in HMECs correlate with misregulation of SIRT1 leading to increased levels of acetylated pRb as well as acetylated H4K16 both globally and at telomeric regions. These results identify a novel form of cellular senescence and provide a potential molecular basis for the rapid cell- and tissue- specific predisposition of breast cancer development associated with BRCA1 haploinsufficiency.

PAF is a five-subunit protein complex composed of Paf1, Cdc73, Leo1, Rtf1 and Ctr9, which was purified from yeast in association with RNA polymerase II and which is believed to function in transcription elongation. However, no direct proof exists for this yet. To assay whether PAF is required in elongation, we determined the in vitro transcription-elongation efficiencies of mutant cell extracts using a DNA template containing two G-less cassettes. paf1D or cdc73D cell extracts showed reduced transcription-elongation efficiencies (16-18% of the wild-type levels), whereas leo1D and rtf1D showed wild-type levels. In vivo transcription efficiency was diminished in the four mutants analysed, as determined by their abilities to transcribe lacZ. Our work provides molecular evidence that PAF has a positive role in transcription elongation and is composed of at least two functionally different types of subunits (Paf1-Cdc73 and Leo1-Rtf1).