Christopher S. Nabel

AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation

AID/APOBEC family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, an important process for transcriptional activation. While oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (mC) or 5-hydroxymethylcytosine (hmC) have emerged as alternatives. Here, we have addressed the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on mC relative to cytosine, their canonical substrate, and no detectable deamination of hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of hmC was undetectable in genomic DNA, while oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation.

HHV-8-negative, idiopathic multicentric Castleman disease: novel insights into biology, pathogenesis, and therapy

Multicentric Castlemans disease (MCD) describes a heterogeneous group of disorders involving proliferation of morphologically benign lymphocytes due to excessive proinflammatory hypercytokinemia, most notably of interleukin-6. Patients demonstrate intense episodes of systemic inflammatory symptoms, polyclonal lymphocyte and plasma cell proliferation, autoimmune manifestations, and organ system impairment. Human herpes virus-8 (HHV-8) drives the hypercytokinemia in all HIV-positive patients and some HIV-negative patients. There is also a group of HIV-negative and HHV-8-negative patients with unknown etiology and pathophysiology, which we propose referring to as idiopathic MCD (iMCD). Here, we synthesize what is known about iMCD pathogenesis, present a new subclassification system, and propose a model of iMCD pathogenesis. MCD should be subdivided into HHV-8-associated MCD and HHV-8-negative MCD or iMCD. The lymphocyte proliferation, histopathology, and systemic features in iMCD are secondary to hypercytokinemia, which can occur with several other diseases. We propose that 1 or more of the following 3 candidate processes may drive iMCD hypercytokinemia: systemic inflammatory disease mechanisms via autoantibodies or inflammatory gene mutations, paraneoplastic syndrome mechanisms via ectopic cytokine secretion, and/or a non-HHV-8 virus. Urgent priorities include elucidating the process driving iMCD hypercytokinemia, identifying the hypercytokine-secreting cell, developing consensus criteria for diagnosis, and building a patient registry to track cases.

Deacetylase-Independent Function of HDAC3 in Transcription and Metabolism Requires Nuclear Receptor Corepressor

Histone deacetylases (HDACs) are believed to regulate gene transcription by catalyzing deacetylation reactions. HDAC3 depletion in mouse liver upregulates lipogenic genes and results in severe hepatosteatosis. Here we show that pharmacologic HDAC inhibition in primary hepatocytes causes histone hyperacetylation but does not upregulate expression of HDAC3 target genes. Meanwhile, deacetylase-dead HDAC3 mutants can rescue hepatosteatosis and repress lipogenic genes expression in HDAC3-depleted mouse liver, demonstrating that histone acetylation is insufficient to activate gene transcription. Mutations abolishing interactions with the nuclear receptor corepressor (NCOR or SMRT) render HDAC3 nonfunctional in vivo. Additionally, liver-specific knockout of NCOR, but not SMRT, causes metabolic and transcriptomal alterations resembling those of mice without hepatic HDAC3, demonstrating that interaction with NCOR is essential for deacetylase-independent function of HDAC3. These findings highlight nonenzymatic roles of a major HDAC in transcriptional regulation in vivo and warrant reconsideration of the mechanism of action of HDAC inhibitors.

Ebolavirus Proteins Suppress the Effects of Small Interfering RNA by Direct Interaction with the Mammalian RNA Interference Pathway

ABSTRACT Cellular RNA interference (RNAi) provides a natural response against viral infection, but some viruses have evolved mechanisms to antagonize this form of antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi by encoding suppressors of RNA silencing (SRSs), we screened all EBOV proteins using an RNAi assay initiated by exogenously delivered small interfering RNAs (siRNAs) against either an EBOV or a reporter gene. In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.

A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples

Eukaryotic microorganisms are important but understudied components of the human microbiome. Here we present a pipeline for analysis of deep sequencing data on single cell eukaryotes. We designed a new 18S rRNA gene-specific PCR primer set and compared a published rRNA gene internal transcribed spacer (ITS) gene primer set. Amplicons were tested against 24 specimens from defined eukaryotes and eight well-characterized human stool samples. A software pipeline https://sourceforge.net/projects/brocc/ was developed for taxonomic attribution, validated against simulated data, and tested on pyrosequence data. This study provides a well-characterized tool kit for sequence-based enumeration of eukaryotic organisms in human microbiome samples.

Demystifying DNA Demethylation

DNA modifying and repair enzymes make a new connection in the mechanism of DNA demethylation. Variability and adaptability are necessary for overcoming the challenges of multicellular life. To address this need, nature has evolved a substantial enzymatic toolbox for altering cytosine within the genome. Methylation of the nucleotide cytosine (C) at the 5-position of the base has profound impacts on gene expression and cellular identity. The reverse of this process, DNA demethylation, is equally important for cleaning the genomic slate during embryogenesis or achieving rapid reactivation of previously silenced genes. Although the mechanism of DNA methylation has been rigorously established, active DNA demethylation in mammals has remained enigmatic, as disparate observations have failed to coalesce into a consistent model. Cytosine deamination, oxidation, and base excision repair enzymes have been proposed in a dizzying variety of combinations (1). Against this backdrop, two reports in this issue, by Ito et al. (2) on page 1300 and He et al. on page 1303 (3), help bring new clarity to the mechanistic model for DNA demethylation.

Clinicopathologic analysis of TAFRO syndrome demonstrates a distinct subtype of HHV‐8‐negative multicentric Castleman disease

Multicentric Castleman disease (MCD) describes a heterogeneous group of disorders involving systemic inflammation, characteristic lymph node histopathology, and multi‐organ dysfunction because of pathologic hypercytokinemia. Whereas Human Herpes Virus‐8 (HHV‐8) drives the hypercytokinemia in a cohort of immunocompromised patients, the etiology of HHV‐8‐negative MCD is idiopathic (iMCD). Recently, a limited series of iMCD cases in Japan sharing a constellation of clinical features, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O) has been described as TAFRO syndrome. Herein, we report clinicopathological findings on 25 patients (14 males and 11 females; 23 Japanese‐born and two US‐born), the largest TAFRO syndrome case series, including the first report of cases from the USA. The median age of onset was 50 years old (range: 23–72). The frequency of each feature was as follows: thrombocytopenia (21/25), anasarca (24/25), fever (21/25), organomegaly (25/25), and reticulin fibrosis (13/16). These patients frequently demonstrated abdominal pain, elevated serum alkaline phosphatase levels, and acute kidney failure. Surprisingly, none of the cases demonstrated marked hypergammoglobulinemia, which is frequently reported in iMCD. Lymph node biopsies revealed atrophic germinal centers with enlarged nuclei of endothelial cells and proliferation of endothelial venules in interfollicular zone. 23 of 25 cases were treated initially with corticosteroids; 12 patients responded poorly and required further therapy. Three patients died during the observation period (median: 9 months) because of disease progression or infections. TAFRO syndrome is a unique subtype of iMCD that demonstrates characteristic clinicopathological findings. Further study to clarify prognosis, pathophysiology, and appropriate treatment is needed. Am. J. Hematol. 91:220–226, 2016.

Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism

Modification of cytosine-guanine dinucleotides (CpGs) is a key part of mammalian epigenetic regulation and helps shape cellular identity. Tet enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) in CpGs to 5-hydroxymethylcytosine (hmC), or onward to 5-formylcytosine (fC) or 5-carboxylcytosine (caC). The multiple mC oxidation products, while intricately linked, are postulated to play independent epigenetic roles, making it critical to understand how the products of stepwise oxidation are established and maintained. Using highly sensitive isotope-based studies, we newly show that Tet2 can yield fC and caC by iteratively acting in a single encounter with mC-containing DNA, without release of the hmC intermediate, and that the modification state of the complementary CpG has little impact on Tet2 activity. By revealing Tet2 as an iterative, de novo mC oxygenase, our study provides insight into how features intrinsic to Tet2 shape the epigenetic landscape.

Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase

Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID’s functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID’s selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2′-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID’s reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2′-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID’s closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2′-fluoro-RNA substrates, AID’s deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID’s DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA.

APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA

Abstract AID/APOBEC family enzymes are best known for deaminating cytosine bases to uracil in single-stranded DNA, with characteristic sequence preferences that can produce mutational signatures in targets such as retroviral and cancer cell genomes. These deaminases have also been proposed to function in DNA demethylation via deamination of either 5-methylcytosine (mC) or TET-oxidized mC bases (ox-mCs), which include 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. One specific family member, APOBEC3A (A3A), has been shown to readily deaminate mC, raising the prospect of broader activity on ox-mCs. To investigate this claim, we developed a novel assay that allows for parallel profiling of activity on all modified cytosines. Our steady-state kinetic analysis reveals that A3A discriminates against all ox-mCs by >3700-fold, arguing that ox-mC deamination does not contribute substantially to demethylation. A3A is, by contrast, highly proficient at C/mC deamination. Under conditions of excess enzyme, C/mC bases can be deaminated to completion in long DNA segments, regardless of sequence context. Interestingly, under limiting A3A, the sequence preferences observed with targeting unmodified cytosine are further exaggerated when deaminating mC. Our study informs how methylation, oxidation, and deamination can interplay in the genome and suggests A3As potential utility as a biotechnological tool to discriminate between cytosine modification states.