Elizabeth M. Duncan

Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF.

Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular ‘effectors’ involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1–15)K4me3 (residues 1–15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1–15)K4me3-bound states. H3(1–15)K4me3 interacts through anti-parallel β-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1–15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin.

ABSTRACT The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.

Expression Patterns and Post-translational Modifications Associated with Mammalian Histone H3 Variants

Covalent histone modifications and the incorporation of histone variants bring about changes in chromatin structure that in turn alter gene expression. Interest in non-allelic histone variants has been renewed, in part because of recent work on H3 (and other) histone variants. However, only in mammals do three non-centromeric H3 variants (H3.1, H3.2, and H3.3) exist. Here, we show that mammalian cell lines can be separated into two different groups based on their expression of H3.1, H3.2, and H3.3 at both mRNA and protein levels. Additionally, the ratio of these variants changes slightly during neuronal differentiation of murine ES cells. This difference in H3 variant expression between cell lines could not be explained by changes in growth rate, cell cycle stages, or chromosomal ploidy, but rather suggests other possibilities, such as changes in H3 variant incorporation during differentiation and tissue- or species-specific H3 variant expression. Moreover, quantitative mass spectrometry analysis of human H3.1, H3.2, and H3.3 showed modification differences between these three H3 variants, suggesting that they may have different biological functions. Specifically, H3.3 contains marks associated with transcriptionally active chromatin, whereas H3.2, in contrast, contains mostly silencing modifications that have been associated with facultative heterochromatin. Interestingly, H3.1 is enriched in both active and repressive marks, although the latter marks are different from those observed in H3.2. Although the biological significance as to why mammalian cells differentially employ three highly similar H3 variants remains unclear, our results underscore potential functional differences between them and reinforce the general view that H3.1 and H3.2 in mammalian cells should not be treated as equivalent proteins.

Cathepsin L Proteolytically Processes Histone H3 During Mouse Embryonic Stem Cell Differentiation

Chromatin undergoes developmentally-regulated structural and chemical changes as cells differentiate, which subsequently lead to differences in cellular function by altering patterns of gene expression. To gain insight into chromatin alterations that occur during mammalian differentiation, we turned to a mouse embryonic stem cell (ESC) model. Here we show that histone H3 is proteolytically cleaved at its N-terminus during ESC differentiation. We map the sites of H3 cleavage and identify Cathepsin L as a protease responsible for proteolytically processing the N-terminal H3 tail. In addition, our data suggest that H3 cleavage may be regulated by covalent modifications present on the histone tail itself. Our studies underscore the intriguing possibility that histone proteolysis, brought about by Cathepsin L and potentially other family members, plays a role in development and differentiation that was not previously recognized.

Localization of the PE methylation pathway and SR-BI to the canalicular membrane evidence for apical PC biosynthesis that may promote biliary excretion of phospholipid and cholesterol

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.

Loci on chromosomes 14 and 2, distinct from ABCG5/ABCG8, regulate plasma plant sterol levels in a C57BL/6J x CASA/Rk intercross

Plasma plant sterol levels differ among humans due to genetic and dietary factors. A disease characterized by high plasma plant sterol levels, β-sitosterolemia, was recently found to be due to mutations at the ABCG5/ABCG8 locus. To detect variants at this and other loci, a genetic cross was carried out between two laboratory mouse strains. Parental C57BL/6J had almost twice the campesterol and sitosterol levels compared with parental CASA/Rk mice, and F1 mice had levels halfway between the parentals. An intercross between F1s was performed and plasma plant sterol levels measured in 102 male and 99 female F2 mice. Plasma plant sterols in F2s displayed a unimodal distribution, suggesting the effects of several rather a single major gene. In the F2 mice, a full genome scan revealed significant linkages on chromosomes 14 and 2. With regard to chromosome 14, analysis showed a single peak for linkage at 17 cM with a logarithm of odds (LOD) score of 9.9, designated plasma plant sterol 14 (Plast14). With regard to chromosome 2, analysis showed two significant peaks for linkage at 18 and 65 cMs with LOD scores of 4.1 and 3.65, respectively, designated Plast2a and Plast2b, respectively. Four interactions between loci, predominantly of an additive nature, were also demonstrated, the most significant between Plast14 and Plast2b (LOD 16.44). No significant linkage or gene interaction was detected for the ABCG5/ABCG8 locus on chromosome 17. Therefore, other genes besides ABCG5/ABCG8 influence plasma plant sterol levels and now become candidates to explain differences in plasma plant sterol levels between humans.

Phytosterolemia on the island of Kosrae founder effect for a novel ABCG8 mutation results in high carrier rate and increased plasma plant sterol levels

Screening of 932 adults on the Pacific island of Kosrae for plasma plant sterol levels disclosed three subjects, two of them asymptomatic, with phytosterolemia. Sequencing the ATP binding cassette subfamily G member 8 (ABCG8) gene revealed a novel exon 2 mutation that causes a change in codon 24 from glutamine to histidine and a frame shift followed by a premature stop codon, precluding the formation of a functional ABCG8 protein. Genotyping of 1,090 Kosraens revealed 150 as carriers, a 13.8% carrier rate. DNA sequencing of 67 carriers revealed the same mutation as in the probands. In carriers, plasma campesterol and sitosterol levels were 55% and 30% higher, respectively, than in noncarriers. Moreover, compared with noncarriers, carriers showed 21% lower plasma levels of lathosterol, a surrogate marker for cholesterol biosynthesis. There was no difference between the groups in plasma total cholesterol, triglycerides, apolipoprotein B, or apolipoprotein A-I levels. In summary, on the island of Kosrae, a strong founder effect of a mutant ABCG8 allele results in a large number of carriers with increased plasma plant sterol levels and decreased lathosterol levels. The latter finding suggests that heterozygosity for a mutated ABCG8 allele results in a modest increase in dietary cholesterol absorption and a decrease in cholesterol biosynthesis.

Set1 and MLL1/2 Target Distinct Sets of Functionally Different Genomic Loci In Vivo

Histone H3 lysine 4 trimethylation (H3K4me3) is known to correlate with both active and poised genomic loci, yet many questions remain regarding its functional roles in vivo. We identify functional genomic targets of two H3K4 methyltransferases, Set1 and MLL1/2, in both the stem cells and differentiated tissue of the planarian flatworm Schmidtea mediterranea. We show that, despite their common substrate, these enzymes target distinct genomic loci in vivo, which are distinguishable by the pattern each enzyme leaves on the chromatin template, i.e., the breadth of the H3K4me3 peak. Whereas Set1 targets are largely associated with the maintenance of the stem cell population, MLL1/2 targets are specifically enriched for genes involved in ciliogenesis. These data not only confirm that chromatin regulation is fundamental to planarian stem cell function but also provide evidence for post-embryonic functional specificity of H3K4me3 methyltransferases in vivo.

Loci controlling plasma non-HDL and HDL cholesterol levels in a C57BL /6J x CASA /Rk intercross.

Plasma non-HDL and HDL cholesterol levels are predictors of cardiovascular diseases. We carried out a genetic cross between two laboratory inbred mouse strains, C57BL/6J and CASA/Rk, to detect loci that control the plasma levels of non-HDL and HDL cholesterol. With regard to non-HDL cholesterol, chow-fed CASA/Rk males and females had 87% and 25% higher levels, respectively, than did C57BL/6Js. The levels of non-HDL cholesterol in F1s were similar to C57BL/6J. There was no strain difference in HDL cholesterol levels. An intercross between F1s was performed, and plasma non-HDL and HDL cholesterol was measured in 185 male and 184 female mice. In both male and female F2 mice, plasma non-HDL and HDL cholesterol levels were unimodally distributed; however, in both cases the values for females were significantly lower than for males. Therefore, linkage analysis was performed with sex as a covariate. Significant linkage for non-HDL cholesterol was found on chromosome 6 at 49 cM (LOD 5.17), chromosome 4 at 55 cM (LOD 4.22), and chromosome 8 at 7 cM (LOD 3.68). Significant linkage for HDL cholesterol was found on chromosome 9 at 14 cM (LOD 7.52) and chromosome 8 at 76 cM (LOD 4.69). A significant epistatic interaction involving loci on chromosomes 2 and 5 was also observed for non-HDL cholesterol. In summary, linkage analysis in these cross-identified novel loci confirmed previously identified loci in control of plasma non-HDL and HDL cholesterol and disclosed a novel interaction in controlling non-HDL cholesterol levels in the mouse.

Two loci on chromosome 9 control bile acid composition : evidence that a strong candidate gene, Cyp8b1, is not the culprit

An intercross between C57BL/6J and CASA/Rk mice was used to study the genetics of biliary bile acid composition. In parental strains, male C57BL/6J mice had significantly higher cholic acid (CA; 14%) and lower β-muricholic acid (βMC; 27%) than CASA/Rk mice, whereas females did not differ. However, quantitative trait locus analysis of F2 mice revealed no significant chromosome 9 loci in males but loci in females on chromosome 9 for percentage CA (%CA) at 72 centimorgan (cM) [logarithm of the odds (LOD) 5.89] and %βMC at 54 cM (LOD 4.09). Chromosome 9 congenic and subcongenic strains representing CASA/Rk intervals 38–73 cM (9KK) and 68–73 cM (9DKK) on the C57BL/6J background were made. In 9KK and 9DKK males, %CA was increased and %βMC was unchanged, whereas in 9KK but not 9DKK females, %CA was increased and %βMC was decreased. Sterol 12α-hydroxylase (Cyp8b1) channels bile acid precursors into CA and maps at chromosome 9 (73 cM). However, there was no significant difference in Cyp8b1 mRNA or enzymatic activity between parental mice, parental-congenic-subcongenic mice, or high-low biliary %CA F2 mice. In summary, two chromosome 9 loci control sexually dimorphic effects on biliary bile acid composition: a distal (68–73 cM) major determinant in males, and a more proximal (38–68 cM) major determinant in females. In this intercross, Cyp8b1, a strong candidate, does not appear to be responsible.