Childhood Maltreatment and Leukocyte Telomere Length: Cardiac Vagal Activity Influences the Relation in Older Adults.

OBJECTIVE: Childhood maltreatment is associated with shorter leukocyte telomere length (LTL). However, the influence of cardiac vagal control on this relation is unknown. We examined whether cardiac vagal control at rest and in response to stress moderates or cross-sectionally mediates the relationship between childhood maltreatment and LTL. METHODS: Participants were 1179 men and women (aged 65 [7.2] years) suffering from coronary artery disease or non-cardiovascular chronic disease. They completed a childhood maltreatment questionnaire and underwent a stress protocol while electrocardiogram was monitored. High-frequency heart rate variability (HF-HRV) measures were obtained at rest, during stress, and after stress in absolute and normalized units (nu). LTL was measured using quantitative polymerase chain reaction. Mediation and moderation analyses were performed. RESULT: HF-HRV and HF-HRV in normalized units (HFnu) measures did not mediate the childhood maltreatment-LTL relation. However, baseline HFnu ( p = .027) and HFnu reactivity ( p = .051) moderated the relation. Specifically, maltreatment was associated with significantly lower LTL among those with baseline HFnu at ( b = -0.059, p = .003) or below the mean ( b = -0.103, p < .001), but not among those with higher baseline HFnu. It was also associated with significantly lower LTL among participants who showed either blunted ( b = -0.058, p = .004) or increased HFnu ( b = -0.099, p = .001) responses to stress but not in those with large decreases in HFnu. CONCLUSIONS: Childhood maltreatment was associated with lower LTL in those who showed a distinct cardiac vagal profile at baseline and in response to stress. The mechanisms and implications remain to be determined.

Childhood maltreatment and leukocyte telomere length in men and women with chronic illness: an evaluation of moderating and mediating influences.

BACKGROUND: Childhood maltreatment can result in lifelong psychological and physical sequelae, including coronary artery disease (CAD). Mechanisms leading to increased risk of illness may involve emotional dysregulation and shortened leukocyte telomere length (LTL). METHODS: To evaluate whether (1) childhood maltreatment is associated with shorter LTL among older adults with CAD or other chronic illnesses; (2) sex and/or CAD status influence these results; and (3) symptoms of anxiety, depression, and stress moderate or mediate the association between childhood maltreatment and LTL, men and women (N = 1247; aged 65 ± 7.2 years) with and without CAD completed validated questionnaires on childhood maltreatment, symptoms of depression, anxiety, and perceived stress. LTL was measured using quantitative polymerase chain reaction. Analyses included bivariate correlations, hierarchical regressions, and moderation/mediation analyses, controlling for sociodemographic and lifestyle variables. RESULTS: Childhood maltreatment was associated with significantly shorter LTL (r = -0.059, p = 0.038, b = -0.016, p = 0.005). This relation was not moderated by depression, anxiety, nor perceived stress, though there was mitigated evidence for absence of a maltreatment-LTL relation in men with CAD. Stress perception (but not anxiety or depression) partially mediated the relation between childhood maltreatment and LTL [Indirect effect, b = -0.0041, s.e. = 0.002, 95% CI (-0.0085 to -0.0002)]. CONCLUSIONS: Childhood maltreatment was associated with accelerated biological aging independently of patient characteristics. Emotional dysregulation resulting in chronic stress may contribute to this process. Whether stress management or other interventions may help prevent or slow premature aging in those who have suffered maltreatment requires study.

NPM and NPM-MLF1 interact with chromatin remodeling complexes and influence their recruitment to specific genes.

Nucleophosmin (NPM1) is frequently mutated or subjected to chromosomal translocation in acute myeloid leukemia (AML). NPM protein is primarily located in the nucleus, but the recurrent NPMc+ mutation, which creates a nuclear export signal, is characterized by cytoplasmic localization and leukemogenic properties. Similarly, the NPM-MLF1 translocation product favors the partial cytoplasmic retention of NPM. Regardless of their common cellular distribution, NPM-MLF1 malignancies engender different effects on hematopoiesis compared to NPMc+ counterparts, highlighting possible aberrant nuclear function(s) of NPM in NPMc+ and NPM-MLF1 AML. We performed a proteomic analysis and found that NPM and NPM-MLF1 interact with various nuclear proteins including subunits of the chromatin remodeling complexes ISWI, NuRD and P/BAF. Accordingly, NPM and NPM-MLF1 are recruited to transcriptionally active or repressed genes along with NuRD subunits. Although the overall gene expression program in NPM knockdown cells is similar to that resulting from NPMc+, NPM-MLF1 expression differentially altered gene transcription regulated by NPM. The abnormal gene regulation imposed by NPM-MLF1 can be characterized by the enhanced recruitment of NuRD to gene regulatory regions. Thus, different mechanisms would orchestrate the dysregulation of NPM function in NPMc+- versus NPM1-MLF1-associated leukemia.

Lineage restriction analyses in CHIP indicate myeloid bias for TET2 and multipotent stem cell origin for DNMT3A.

We analyzed DNA from polymorphonuclear (PMN) cells, monocytes, B cells, and T cells of 107 individuals with clonal hematopoiesis of indeterminate potential (CHIP) to perform lineage restriction analysis of different gene mutations. Three lineage categories were defined: myeloid (PMN with or without monocytes), myelolympho-B (myeloid and B cells), and multipotent (myeloid, B and T cells). Six individuals with aberrant patterns were excluded from analysis. Ninety-four had a single mutation (56 in DNMT3A, 24 in TET2, 7 in other genes [JAK2, ASXL1, CBL or TP53]). Fourteen had multiple mutations. The lineage restriction patterns of single DNMT3A- or TET2-mutated individuals were different. The proportion of myeloid restricted mutations was higher for TET2 (54.2%, 13 of 24) than for DNMT3A (23.2%, 13 of 56) (P < .05). It was similar for myelolympho-B category but with a 1.5 fold greater proportion of myeloid cells for TET2 individuals (P < .05). Importantly, 0% (0 of 24) of the individuals with TET2 mutation in the multipotent category in contrast to 35.7% (20 of 56) for DNMT3A (P < .01). The clone size predicted multipotent pattern for DNMT3A suggesting a time delay for extensive lineage clonal dominance. These distinctive features may be important in deciphering the transformation mechanisms of these frequent mutations.

DNMT3A and TET2 dominate clonal hematopoiesis and demonstrate benign phenotypes and different genetic predispositions.

Age-associated clonal hematopoiesis caused by acquired mutations in myeloid cancer-associated genes is highly prevalent in the normal population. Its etiology, biological impact on hematopoiesis, and oncogenic risk is poorly defined at this time. To gain insight into this phenomenon, we analyzed a cohort of 2530 related and unrelated hematologically normal individuals (ages 55 to 101 years). We used a sensitive gene-targeted deep sequencing approach to gain precision on the exact prevalence of driver mutations and the proportions of affected genes. Mutational status was correlated with biological parameters. We report a higher overall prevalence of driver mutations (13.7%), which occurred mostly (93%) in DNMT3A or TET2 and were highly age-correlated. Mutation in these 2 genes had some distinctive effects on end points. TET2 mutations were more age-dependent, associated with a modest neutropenic effect (9%, P = .012), demonstrated familial aggregation, and associated with chronic obstructive pulmonary disease. Mutations in DNMT3A had no impact on blood counts or indices. Mutational burden of both genes correlated with X-inactivation skewing but no significant association with age-adjusted telomere length reduction was documented. The discordance between the high prevalence of mutations in these 2 genes and their limited biological impact raise the question of the potential role of dysregulated epigenetic modifiers in normal aging hematopoiesis, which may include support to failing hematopoiesis.

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik(NULL) hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

Ikaros interacts with P-TEFb and cooperates with GATA-1 to enhance transcription elongation.

Ikaros is associated with both gene transcriptional activation and repression in lymphocytes. Ikaros acts also as repressor of human γ-globin (huγ-) gene transcription in fetal and adult erythroid cells. Whether and eventually, how Ikaros can function as a transcriptional activator in erythroid cells remains poorly understood. Results presented herein demonstrate that Ikaros is a developmental-specific activator of huγ-gene expression in yolk sac erythroid cells. Molecular analysis in primary cells revealed that Ikaros interacts with Gata-1 and favors Brg1 recruitment to the human ß-globin Locus Control Region and the huγ-promoters, supporting long-range chromatin interactions between these regions. Additionally, we demonstrate that Ikaros contributes to transcription initiation and elongation of the huγ-genes, since it is not only required for TBP and RNA Polymerase II (Pol II) assembly at the huγ-promoters but also for conversion of Pol II into the elongation-competent phosphorylated form. In agreement with the latter, we show that Ikaros interacts with Cyclin-dependent kinase 9 (Cdk9), which contributes to efficient transcription elongation by phosphorylating the C-terminal domain of the large subunit of Pol II on Serine 2, and favours Cdk9 recruitment to huγ-promoters. Our results show that Ikaros exerts dual functionality during gene activation, by promoting efficient transcription initiation and elongation.

Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters.

Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human beta-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the beta-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual beta-like globin genes. Moreover, in transgenic mice expressing the entire human beta-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter beta-like globin gene expression. Here, we show that abnormal expression of human beta-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human epsilon- and gamma-promoters, but not at the beta-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human gamma-promoters. We also find that HS2 is dispensable for high-level human beta-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.

The associations of hostility and defensiveness with telomere length are influenced by sex and health status.

BACKGROUND: Shorter telomere length (TL) may indicate premature cellular aging and increased risk for disease. While there is substantial evidence for shorter TL in individuals suffering from psychiatric disorders, data is scarce on maladaptive personality traits related to coronary artery disease (CAD). The purpose of this study was to evaluate the association of TL with hostility and defensiveness in individuals with CAD or other non-cardiovascular illnesses and whether associations were moderated by CAD status and sex. METHODS: One thousand thirty-six individuals (Mage = 65.40 ± 6.73 years) with and without CAD completed the Marlowe-Crowne Social Desirability Scale and the Cook-Medley Hostility Scale. Relative TL was measured via quantitative polymerase chain reaction of total genomic DNA samples. Analyses involved hierarchical regressions on TL, performed separately for hostility and defensiveness, controlling for pertinent sociodemographic, behavioural, and medical risk factors. Separate analyses were performed on 25 healthy participants. RESULTS: A hostility by sex interaction emerged (ß = - .08, p = .006) in the patient groups, where greater hostility was associated with shorter TL in women only (p < .01). A Defensiveness by CAD status interaction (ß = - .06, p = .049) revealed longer TL in more defensive CAD patients only (p = .06). In healthy men, shorter TL was observed in those with greater defensiveness (ß = .52, p = .006) but lower hostility (ß = - .43, p = .049). CONCLUSION: Hostility and defensiveness are differentially associated with TL as a function of sex and health status. The implication of these results for health remains to be determined, but propose an additional pathway through which the effect of maladaptive personality traits may contribute to CV and other disease.

MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells.

The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.

High-sensitivity C-reactive protein is associated with clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis of indeterminate potential (CHIP) is predictive of hematological cancers and cardiovascular diseases, but the etiology of CHIP initiation and clonal expansion is unknown. Several lines of evidence suggest that proinflammatory cytokines may favor mutated hematopoietic stem cell expansion. To investigate the potential link between inflammation and CHIP, we performed targeted deep sequencing of 11 genes previously implicated in CHIP in 1887 subjects aged >70 years from the Montreal Heart Institute Biobank, of which 1359 had prior coronary artery disease (CAD), and 528 controls did not. We assessed association of CHIP with log transformed high-sensitivity C-reactive protein (hs-CRP), a validated biomarker of inflammation. CHIP was identified in 427 of the 1887 subjects (22.6%). CHIP mutations were more frequently identified in DNMT3A (11.6%) and TET2 (6.1%), with a higher proportion of TET2 mutations occurring in controls than in patients with CAD (9.0% vs 4.9%, P < .001). CHIP carriers had 21% higher hs-CRP levels compared with their noncarrier counterparts (eß = 1.21, 95% confidence interval [CI]: 1.08 to 1.36; P = .001). A similar effect was observed in the subgroup of patients with known CAD (eß = 1.22, 95% CI: 1.06 to 1.41; P = .005). These findings confirm the association between inflammation and CHIP. This association may open investigational avenues aimed at documenting mechanisms linking inflammation to clonal progression and ultimately supports prevention interventions to attenuate CHIP's impact on cardiovascular disease and cancer.

Structure of Pb-rich chabournéite from Jas Roux, France.

The crystal structure of a specimen of `Pb-rich' chabournéite from Jas Roux, Hautes-Alpes, France, with the chemical formula obtained by electron microprobe analysis of Ag(0.04 (1))Tl(2.15 (2))Pb(0.64 (1))Sb(5.12 (1))As(5.05 (1))S(17.32 (5)), has been solved by X-ray single-crystal diffraction on the basis of 36,550 observed reflections (with F(o) > 4σF(o)) with a final R1 = 0.074. Pb-rich chabournéite is triclinic P1, with unit-cell parameters a = 8.5197 (4), b = 42.461 (2), c = 16.293 (8) Å, α = 83.351 (2), ß = 90.958 (2), γ = 84.275 (2)°, V = 5823 (3) Å(3). Its structural formula is close to [Tl2(Pb(0.8)Tl(0.1)Sb(1.1))](Sb(4.1)As(4.9))S17, with Z = 8. Its crystal structure is formed by the alternation of two pairs of slabs along the b axis, deriving from the SnS and PbS archetypes, respectively. 104 independent cation sites and 136 S sites occur in the unit cell. Slab interfaces show the alternation, along c, of Tl sites, ninefold coordinated, with Pb, Sb or mixed/split (Pb,Sb) and (Pb,Tl) sites. Within the slabs, 72 independent M(3+) sites (M(3+) = As, Sb) occur. Considering M(3+)-S bond distances shorter than 2.70 Å, MS3 triangular pyramidal groups are condensed according to various M(m)S(n) chain fragments (`polymers'). The solution of the crystal structure of chabournéite allows its comparison with the closely related homeotypes protochabournéite and dalnegroite.

Direct protein interactions are responsible for Ikaros-GATA and Ikaros-Cdk9 cooperativeness in hematopoietic cells.

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

Ikaros and GATA-1 combinatorial effect is required for silencing of human gamma-globin genes.

During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human beta-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human gamma-gene promoters, thereby contributing to gamma-globin gene silencing at the time of the gamma- to beta-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human gamma-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.

Onset and inheritance of abnormal epigenetic regulation in hematopoietic cells.

Abnormal epigenetic regulation of gene expression contributes significantly to a variety of human pathologies including cancer. Deletion of hypersensitive site 2 (HS2) at the human beta-globin locus control region can lead to abnormal epigenetic regulation of globin genes in transgenic mice. Here, two HS2-deleted transgenic mouse lines were used as model to demonstrate that heritable alteration of chromatin organization at the human beta-globin locus in multipotent hematopoietic progenitors contributes to the abnormal expression of the beta-globin gene in mature erythroid cells. This alteration is characterized by specific patterns of histone covalent modifications that are inherited during erythropoiesis and, moreover, is plastic because it can be reverted by transient treatment with the histone deacetylase inhibitor Trichostatin A. Altogether, our results indicate that aberrant epigenetic regulation can be detected and modified before tissue-specific gene transcription, a finding which may lead to novel strategies for the prevention of chromatin-related pathologies.