Jeremy P. Brown

Mild head injury, anticoagulants, and risk of intracranial injury

We studied intracranial damage in patients with mild head injuries who were taking warfarin. Of the 215,785 individuals who visited the Mount Auburn and Beth Israel accident and emergency departments during our study, we identified records for 144 patients by anticoagulation status and computed tomography (CT) imaging. We retrospectively reviewed these patients and ten (7%, 95% CI 3-11) with clinically important injuries on cranial CT. Our findings suggest that patients with head injuries who receive anticoagulants have a similar or greater risk of intracranlal injury to those falling into a previously defined moderate-risk category, invalidating a previous conclusion that CT scanning is unnecessary in such patients.

HP1γ function is required for male germ cell survival and spermatogenesis

BackgroundHP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1α, HP1β and HP1γ, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1γ using a gene-targeted mouse.ResultsWhile targeting the Cbx3 gene (encoding the HP1γ protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1γ protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypoanimals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypotestes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypogerm cells.ConclusionsThe Cbx3 gene product (the HP1γ protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypospermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1γ in transposon silencing and parental imprinting.

Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination

Development of efficient short-term gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study, we present a highly efficient, nonviral non-DNA technology for genetic loading of mouse ES cells based on electroporation of defined mRNA. Here, we show that mouse ES cells can be efficiently loaded with mRNA encoding a green fluorescent reporter protein, resulting in a level of at least 90% of transgene expression without loss of cell viability and phenotype. To show that transgenes, introduced by mRNA electroporation, exert a specific cellular function in transfected cells, we electroporated stably transfected ES cell lines with mRNA encoding FLPe or Cre recombinase proteins in order to excise an FRT- or LoxP-flanked reporter gene. The results, as determined by the disappearance and/or appearance of a fluorescent reporter gene expression, show that FLPe and Cre recombinase proteins, introduced by mRNA electroporation, efficiently exert their function without influence on further culture of undifferentiated ES cell populations and their ability to differentiate towards a specific lineage.

Mammalian HP1 isoforms have specific roles in heterochromatin structure and organization

HP1 is a structural component of heterochromatin. Mammalian HP1 isoforms HP1α, HP1β, and HP1γ play different roles in genome stability, but their precise role in heterochromatin structure is unclear. Analysis of Hp1α-/-, Hp1β-/-, and Hp1γ-/- MEFs show that HP1 proteins have both redundant and unique functions within pericentric heterochromatin (PCH) and also act globally throughout the genome. HP1α confines H4K20me3 and H3K27me3 to regions within PCH, while its absence results in a global hyper-compaction of chromatin associated with a specific pattern of mitotic defects. In contrast, HP1β is functionally associated with Suv4-20h2 and H4K20me3, and its loss induces global chromatin decompaction and an abnormal enrichment of CTCF in PCH and other genomic regions. Our work provides insight into the roles of HP1 proteins in heterochromatin structure and genome stability.

An HP1 isoform-specific feedback mechanism regulates Suv39h1 activity under stress conditions

ABSTRACT The presence of H3K9me3 and heterochromatin protein 1 (HP1) are hallmarks of heterochromatin conserved in eukaryotes. The spreading and maintenance of H3K9me3 is effected by the functional interplay between the H3K9me3-specific histone methyltransferase Suv39h1 and HP1. This interplay is complex in mammals because the three HP1 isoforms, HP1α, β, and γ, are thought to play a redundant role in Suv39h1-dependent deposition of H3K9me3 in pericentric heterochromatin (PCH). Here, we demonstrate that despite this redundancy, HP1α and, to a lesser extent, HP1γ have a closer functional link to Suv39h1, compared to HP1β. HP1α and γ preferentially interact in vivo with Suv39h1, regulate its dynamics in heterochromatin, and increase Suv39h1 protein stability through an inhibition of MDM2-dependent Suv39h1-K87 polyubiquitination. The reverse is also observed, where Suv39h1 increases HP1α stability compared HP1β and γ. The interplay between Suv39h1 and HP1 isoforms appears to be relevant under genotoxic stress. Specifically, loss of HP1α and γ isoforms inhibits the upregulation of Suv39h1 and H3K9me3 that is observed under stress conditions. Reciprocally, Suv39h1 deficiency abrogates stress-dependent upregulation of HP1α and γ, and enhances HP1β levels. Our work defines a specific role for HP1 isoforms in regulating Suv39h1 function under stress via a feedback mechanism that likely regulates heterochromatin formation.

A hypomorphic Cbx3 allele causes prenatal growth restriction and perinatal energy homeostasis defects

ABSTRACTMammals have three HP1 protein isotypes HP1β (CBX1), HP1γ (CBX3) and HP1α (CBX5) that are encoded by the corresponding genes Cbx1, Cbx3 and Cbx5. Recent work has shown that reduction of CBX3 protein in homozygotes for a hypomorphic allele (Cbx3hypo) causes a severe postnatal mortality with around 99% of the homozygotes dying before weaning. It is not known what the causes of the postnatal mortality are. Here we show that Cbx3hypo/hypo conceptuses are significantly reduced in size and the placentas exhibit a haplo-insufficiency. Late gestation Cbx3hypo/hypo placentas have reduced mRNA transcripts for genes involved in growth regulation, amino acid and glucose transport. Blood vessels within the Cbx3hypo/hypo placental labyrinth are narrower than wild-type. Newborn Cbx3hypo/hypo pups are hypoglycemic, the livers are depleted of glycogen reserves and there is almost complete loss of stored lipid in brown adipose tissue (BAT). There is a 10-fold reduction in expression of the BAT-specific Ucp1 gene, whose product is responsible for non-shivering themogenesis. We suggest that it is the small size of the Cbx3hypo/hypo neonates, a likely consequence of placental growth and transport defects, combined with a possible inability to thermoregulate that causes the severe postnatal mortality.

Composite cis-acting epigenetic switches in eukaryotes: lessons from Drosophila Fab-7 for the Igf2-H19 imprinted domain.

One of the central problems of eukaryotic gene regulation is to understand the mechanism(s) by which the activity of enhancer elements is circumscribed such that they only act upon their cognate promoter sequences. Studies on the bithorax gene complex (BX-C) in Drosophila have highlighted the potential problem of enhancer promiscuity and detailed molecular and genetic analyses are now providing insight into how this gene complex resolves the problem through the activity of boundary/silencer elements that can block the communication between enhancers and promoters. Analysis of the mouse Igf2–H19 imprinted locus also suggests a role for boundary/silencer elements, but in this case these elements are invoked to account for the preferential expression of Igf2 and H19 from the paternally and maternally inherited chromosomes respectively despite the presence of functional downstream enhancers. We discuss recent work that has illuminated both of these systems and consider what parallels exist between them.

Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

BackgroundHeterochromatin, which is formed when tri-methyl lysine 9 of histone H3 (H3K9me3) is bound by heterochromatin 1 proteins (HP1s), plays an important role in differentiation and senescence by silencing cell cycle genes. Cardiac myocytes (CMs) accumulate heterochromatin during differentiation and demethylation of H3K9me3 inhibits cell cycle gene silencing and cell cycle exit in CMs; however, it is unclear if this process is mediated by HP1s. In this study, we created a conditional CM-specific HP1 gamma (HP1γ) knockout (KO) mouse model and tested whether HP1γ is required for cell cycle gene silencing and cardiac growth.ResultsHP1γ KO mice were generated by crossing HP1γ floxed mice (fl) with mice expressing Cre recombinase driven by the Nkx2.5 (cardiac progenitor gene) promoter (Cre). We confirmed that deletion of critical exons of HP1γ led to undetectable levels of HP1γ protein in HP1γ KO (Cre;fl/fl) CMs. Analysis of cardiac size and function by echo revealed no significant differences between HP1γ KO and control (WT, Cre, fl/fl) mice. No significant difference in expression of cell cycle genes or cardiac-specific genes was observed. Global transcriptome analysis demonstrated a very moderate effect of HP1γ deletion on global gene expression, with only 51 genes differentially expressed in HP1γ KO CMs. We found that HP1β protein, but not HP1α, was significantly upregulated and that subnuclear localization of HP1β to perinuclear heterochromatin was increased in HP1γ KO CMs. Although HP1γ KO had no effect on H3K9me3 levels, we found a significant reduction in another major heterochromatin mark, tri-methylated lysine 20 of histone H4 (H4K20me3).ConclusionsThese data indicate that HP1γ is dispensable for cell cycle exit and normal cardiac growth but has a significant role in maintaining H4K20me3 and regulating a limited number of genes in CMs.