Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In ...animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungus
, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen of
deletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found the
homolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinct
ISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway in
, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.
Pax gene haploinsufficiency causes a variety of congenital defects. Renal-coloboma syndrome, resulting from mutations in Pax2, is characterized by kidney hypoplasia, optic nerve malformation, and ...hearing loss. Although this underscores the importance of Pax gene dosage in normal development, how differential levels of these transcriptional regulators affect cell differentiation and tissue morphogenesis is still poorly understood. We show that differential levels of zebrafish Pax2a and Pax8 modulate commitment and behavior in cells that eventually contribute to the otic vesicle and epibranchial placodes. Initially, a subset of epibranchial placode precursors lie lateral to otic precursors within a single Pax2a/8-positive domain; these cells subsequently move to segregate into distinct placodes. Using lineage-tracing and ablation analyses, we show that cells in the Pax2a/8+ domain become biased towards certain fates at the beginning of somitogenesis. Experiments involving either Pax2a overexpression or partial, combinatorial Pax2a and Pax8 loss of function reveal that high levels of Pax favor otic differentiation whereas low levels increase cell numbers in epibranchial ganglia. In addition, the Fgf and Wnt signaling pathways control Pax2a expression: Fgf is necessary to induce Pax2a, whereas Wnt instructs the high levels of Pax2a that favor otic differentiation. Our studies reveal the importance of Pax levels during sensory placode formation and provide a mechanism by which these levels are controlled.
We are not so special Lewis, Zachary R; Dunn, Casey W
eLife,
07/2018, Volume:
7
Journal Article
Peer reviewed
Open access
New sequence data from choanoflagellates improves our understanding of the genetic changes that occurred along the branch of the evolutionary tree that gave rise to animals.
ChIP-Seq Analysis in Neurospora crassa Ferraro, Aileen R; Lewis, Zachary A
Methods in molecular biology (Clifton, N.J.),
2018, Volume:
1775
Journal Article
Chromatin immunoprecipitation paired with next-generation sequencing (ChIP-seq) can be used to determine genome-wide distribution of transcriptions factors, transcriptional machinery, or histone ...modifications. DNA-protein interactions are covalently cross-linked with the addition of formaldehyde. Chromatin is prepared and sheared, then immunoprecipitated with the appropriate antibody. After reversal of cross-linking and treating with protease, the resulting DNA fragments are sequenced and mapped to the reference genome to determine overall enrichment. Here we describe a method of ChIP-seq for investigating protein-DNA interactions in the filamentous fungus Neurospora crassa.
Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins and ...modifications of histones and DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation (5mC), methylation of histone H3 lysine 9 (H3K9me), and heterochromatin protein 1 (HP1), and is a model to investigate heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3, and H3K4me2 at 100 bp resolution and explored their interplay. HP1, H3K9me3, and 5mC were extensively co-localized and defined 44 heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation. Interestingly, the centromere was found in an approximately 350 kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation in N. crassa. In contrast, we found that localization of H3K9me3 was independent of 5mC or HP1 at virtually all heterochromatin regions. In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the H3K9 methylation machinery. These data show that A:T-rich RIP'd DNA efficiently directs methylation of H3K9, which in turn, directs methylation of associated cytosines.
The development of whole-genome bisulfite sequencing (WGBS) has resulted in a number of exciting discoveries about the role of DNA methylation leading to a plethora of novel testable hypotheses. ...Methods for constructing sodium bisulfite-converted and amplified libraries have recently advanced to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of reads from methylated DNA in WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. It is especially critical for studies that seek to accurately quantify DNA methylation levels in populations that may segregate for allelic DNA methylation states.
To report our initial experience with the extra-tunical grafting (ETG) procedure. This procedure was recently introduced by UCSF investigators as a tunica-sparing technique for management of penile ...concavity deformities.
We retrospectively reviewed records of patients who underwent ETG at our tertiary-care referral center between 2017 – 2020. A collagen graft made from bovine pericardium (Lyoplant) was placed overlying the defect without violating the tunica albuginea or mobilizing the neurovascular bundle. The stretched penile length (SPL) and circumference at the location of deformity were measured intra-operatively. Patient reported outcomes were evaluated by an anonymous 10-question online survey.
19 men underwent ETG with a median follow-up of 59 (IQR: 24 – 708) days. ETG was performed via either a window (15/19, 78%) or a de-gloving (4/19, 21%) incision with concomitant penile plication performed in 16/19 (84%) patients. Penile circumference increased by an average of 1.4 cm + 0.5 (P = 0.03) at the location of deformity, while pre- and post-operative SPL were similar (14.0 + 1.4 vs 14.0 + 1.3 cm, P = 0.95). Overall patient satisfaction was reported by 13/15 (86%) patients. Twelve out of 15 (80%) patients reported concavity deformity to be “improved”, with 73% reporting “much better”. Among 8 patients with follow up greater than six months, graft palpability was reported in 4/8 (50%) patients but was not bothersome.
The ETG procedure appears to be safe and effective for the treatment of penile concavity deformities. Patient outcomes and satisfaction are favorable at intermediate follow up.
DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair ...(BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.
,
, and
cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these ...life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2's mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of
,
, and
than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases.
Invasive fungal diseases caused by
,
, and
have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to
,
, and
than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.
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