Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands.

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Chromatin Remodelers in the 3D Nuclear Compartment.

Chromatin remodeling complexes (CRCs) use ATP hydrolysis to maintain correct expression profiles, chromatin stability, and inherited epigenetic states. More than 20 CRCs have been described to date, which encompass four large families defined by their ATPase subunits. These complexes and their subunits are conserved from yeast to humans through evolution. Their activities depend on their catalytic subunits which through ATP hydrolysis provide the energy necessary to fulfill cellular functions such as gene transcription, DNA repair, and transposon silencing. These activities take place at the first levels of chromatin compaction, and CRCs have been recognized as essential elements of chromatin dynamics. Recent studies have demonstrated an important role for these complexes in the maintenance of higher order chromatin structure. In this review, we present an overview of the organization of the genome within the cell nucleus, the different levels of chromatin compaction, and importance of the architectural proteins, and discuss the role of CRCs and how their functions contribute to the dynamics of the 3D genome organization.

Insights into HP1a-Chromatin Interactions.

Understanding the packaging of DNA into chromatin has become a crucial aspect in the study of gene regulatory mechanisms. Heterochromatin establishment and maintenance dynamics have emerged as some of the main features involved in genome stability, cellular development, and diseases. The most extensively studied heterochromatin protein is HP1a. This protein has two main domains, namely the chromoshadow and the chromodomain, separated by a hinge region. Over the years, several works have taken on the task of identifying HP1a partners using different strategies. In this review, we focus on describing these interactions and the possible complexes and subcomplexes associated with this critical protein. Characterization of these complexes will help us to clearly understand the implications of the interactions of HP1a in heterochromatin maintenance, heterochromatin dynamics, and heterochromatin's direct relationship to gene regulation and chromatin organization.

Molecular effects of dADD1 misexpression in chromatin organization and transcription.

BACKGROUND: dADD1 and dXNP proteins are the orthologs in Drosophila melanogaster of the ADD and SNF2 domains, respectively, of the ATRX vertebrate's chromatin remodeler, they suppress position effect variegation phenotypes and participate in heterochromatin maintenance. RESULTS: We performed a search in human cancer databases and found that ATRX protein levels were elevated in more than 4.4% of the samples analyzed. Using the Drosophila model, we addressed the effects of over and under-expression of dADD1 proteins in polytene cells. Elevated levels of dADD1 in fly tissues caused different phenotypes, such as chromocenter disruption and loss of banding pattern at the chromosome arms. Analyses of the heterochromatin maintenance protein HP1a, the dXNP ATPase and the histone post-translational modification H3K9me3 revealed changes in their chromatin localization accompanied by mild transcriptional defects of genes embedded in heterochromatic regions. Furthermore, the expression of heterochromatin embedded genes in null dadd1 organisms is lower than in the wild-type conditions. CONCLUSION: These data indicate that dADD1 overexpression induces chromatin changes, probably affecting the stoichiometry of HP1a containing complexes that lead to transcriptional and architectural changes. Our results place dADD1 proteins as important players in the maintenance of chromatin architecture and heterochromatic gene expression.

Ecdysone-Induced 3D Chromatin Reorganization Involves Active Enhancers Bound by Pipsqueak and Polycomb.

Evidence suggests that Polycomb (Pc) is present at chromatin loop anchors in Drosophila. Pc is recruited to DNA through interactions with the GAGA binding factors GAF and Pipsqueak (Psq). Using HiChIP in Drosophila cells, we find that the psq gene, which has diverse roles in development and tumorigenesis, encodes distinct isoforms with unanticipated roles in genome 3D architecture. The BR-C, ttk, and bab domain (BTB)-containing Psq isoform (PsqL) colocalizes genome-wide with known architectural proteins. Conversely, Psq lacking the BTB domain (PsqS) is consistently found at Pc loop anchors and at active enhancers, including those that respond to the hormone ecdysone. After stimulation by this hormone, chromatin 3D organization is altered to connect promoters and ecdysone-responsive enhancers bound by PsqS. Our findings link Psq variants lacking the BTB domain to Pc-bound active enhancers, thus shedding light into their molecular function in chromatin changes underlying the response to hormone stimulus.

Transregulation of microRNA miR-21 promoter by AP-1 transcription factor in cervical cancer cells.

BACKGROUND: Gene expression profiles have demonstrated that miR-21 expression is altered in almost all types of cancers and it has been classified as an oncogenic microRNA. Persistent HPV infection is the main etiologic agent in cervical cancer and induces genetic instability, including disruption of microRNA gene expression. In the present study, we analyzed the underlying mechanism of how AP-1 transcription factor can active miR-21 gene expression in cervical cancer cells. METHODS: To identify that c-Fos and c-Jun regulate the expression of miR-21 we performed RT-qPCR and western blot assays. We analyzed the interaction of AP-1 with miR-21 promoter by EMSA and ChIP assays and determined the mechanism of its regulation by reporter construct plasmids. We identified the nuclear translocation of c-Fos and c-Jun by immunofluorescence microscopy assays. RESULTS: We demonstrated that c-Fos and c-Jun proteins are expressed and regulate the expression of miR-21 in cervical cancer cells. DNA sequence analysis revealed the presence of AP-1 DNA-binding sites in the human miR-21 promoter region. EMSA analyses confirmed the interactions of the miR-21 upstream transcription factor AP-1. ChIP assays further showed the binding of c-Fos to AP-1 sequences from the miR-21 core promoter in vivo. Functional analysis of AP-1 sequences of miR-21 in reporter plasmids demonstrated that these sequences increase the miR-21 promoter activation. CONCLUSIONS: Our findings suggest a physical interaction and functional cooperation between AP-1 transcription factor in the miR-21 promoter and may explain the effect of AP-1 on miR-21 gene expression in cervical cancer cells.

The Dmp8-Dmp18 bicistron messenger RNA enables unusual translation during cellular stress.

Alternatives to the cap mechanism in translation are often used by viruses and cells to allow them to synthesize proteins in events of stress and viral infection. In Drosophila there are hundreds of polycistronic messenger RNA (mRNA), and various mechanisms are known to achieve this. However, proteins in a same mRNA often work in the same cellular mechanism, this is not the case for Drosophila's Swc6/p18Hamlet homolog Dmp18, part of the SWR1 chromatin remodeling complex, who is encoded in a bicistronic mRNA next to Dmp8 (Dmp8-Dmp18 transcript), a structural component of transcription factor TFIIH. The organization of these two genes as a bicistron is conserved in all arthropods, however the length of the intercistronic sequence varies from more than 90 to 2 bases, suggesting an unusual translation mechanism for the second open reading frame. We found that even though translation of Dmp18 occurs independently from that of Dmp8, it is necessary for Dmp18 to be in that conformation to allow its correct translation during cellular stress caused by damage via heat-shock and UV radiation.

dAdd1 and dXNP prevent genome instability by maintaining HP1a localization at Drosophila telomeres.

Telomeres are important contributors to genome stability, as they prevent linear chromosome end degradation and contribute to the avoidance of telomeric fusions. An important component of the telomeres is the heterochromatin protein 1a (HP1a). Mutations in Su(var)205, the gene encoding HP1a in Drosophila, result in telomeric fusions, retrotransposon regulation loss and larger telomeres, leading to chromosome instability. Previously, it was found that several proteins physically interact with HP1a, including dXNP and dAdd1 (orthologues to the mammalian ATRX gene). In this study, we found that mutations in the genes encoding the dXNP and dAdd1 proteins affect chromosome stability, causing chromosomal aberrations, including telomeric defects, similar to those observed in Su(var)205 mutants. In somatic cells, we observed that dXNP and dAdd1 participate in the silencing of the telomeric HTT array of retrotransposons, preventing anomalous retrotransposon transcription and integration. Furthermore, the lack of dAdd1 results in the loss of HP1a from the telomeric regions without affecting other chromosomal HP1a binding sites; mutations in dxnp also affected HP1a localization but not at all telomeres, suggesting a specialized role for dAdd1 and dXNP proteins in locating HP1a at the tips of the chromosomes. These results place dAdd1 as an essential regulator of HP1a localization and function in the telomere heterochromatic domain.

Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation.

Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. Here, we show that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, we found that TFIIH stability is not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients.

Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells.

Tristetraprolin (TTP) is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα), which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR), glucocorticoid receptor (GR) and androgen receptor (AR). In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.

Characterization of the Drosophila group ortholog to the amino-terminus of the alpha-thalassemia and mental retardation X-Linked (ATRX) vertebrate protein.

The human ATRX gene encodes hATRX, a chromatin-remodeling protein harboring an helicase/ATPase and ADD domains. The ADD domain has two zinc fingers that bind to histone tails and mediate hATRX binding to chromatin. dAtrx, the putative ATRX homolog in Drosophila melanogaster, has a conserved helicase/ATPase domain but lacks the ADD domain. A bioinformatic search of the Drosophila genome using the human ADD sequence allowed us to identify the CG8290 annotated gene, which encodes three ADD harboring- isoforms generated by alternative splicing. This Drosophila ADD domain is highly similar in structure and in the amino acids which mediate the histone tail contacts to the ADD domain of hATRX as shown by 3D modeling. Very recently the CG8290 annotated gene has been named dadd1. We show through pull-down and CoIP assays that the products of the dadd1 gene interact physically with dAtrxL and HP1a and all of them mainly co-localize in the chromocenter, although euchromatic localization can also be observed through the chromosome arms. We confirm through ChIP analyses that these proteins are present in vivo in the same heterochromatic regions. The three isoforms are expressed throughout development. Flies carrying transheterozygous combinations of the dadd1 and atrx alleles are semi-viable and have different phenotypes including the appearance of melanotic masses. Interestingly, the dAdd1-b and c isoforms have extra domains, such as MADF, which suggest newly acquired functions of these proteins. These results strongly support that, in Drosophila, the atrx gene diverged and that the dadd1-encoded proteins participate with dAtrx in some cellular functions such as heterochromatin maintenance.

Physical and functional interactions between Drosophila homologue of Swc6/p18Hamlet subunit of the SWR1/SRCAP chromatin-remodeling complex with the DNA repair/transcription factor TFIIH.

The multisubunit DNA repair and transcription factor TFIIH maintains an intricate cross-talk with different factors to achieve its functions. The p8 subunit of TFIIH maintains the basal levels of the complex by interacting with the p52 subunit. Here, we report that in Drosophila, the homolog of the p8 subunit (Dmp8) is encoded in a bicistronic transcript with the homolog of the Swc6/p18(Hamlet) subunit (Dmp18) of the SWR1/SRCAP chromatin remodeling complex. The SWR1 and SRCAP complexes catalyze the exchange of the canonical histone H2A with the H2AZ histone variant. In eukaryotic cells, bicistronic transcripts are not common, and in some cases, the two encoded proteins are functionally related. We found that Dmp18 physically interacts with the Dmp52 subunit of TFIIH and co-localizes with TFIIH in the chromatin. We also demonstrated that Dmp18 genetically interacts with Dmp8, suggesting that a cross-talk might exist between TFIIH and a component of a chromatin remodeler complex involved in histone exchange. Interestingly, our results also show that when the level of one of the two proteins is decreased and the other maintained, a specific defect in the fly is observed, suggesting that the organization of these two genes in a bicistronic locus has been selected during evolution to allow co-regulation of both genes.

Drosophila DREF acting via the JNK pathway is required for thorax development.

The Drosophila Jun N-terminal kinase (JNK) gene basket (bsk) promoter contains a DNA replication-related element (DRE)-like sequence, raising the possibility of regulation by the DNA replication-related element-binding factor (DREF). Chromatin immunoprecipitation assays with anti-DREF IgG showed the bsk gene promoter region to be effectively amplified. Luciferase transient expression assays revealed the DRE-like sequence to be important for bsk gene promoter activity, and knockdown of DREF decreased the bsk mRNA level and the bsk gene promoter activity. Furthermore, knockdown of DREF in the notum compartment of wing discs by pannier-GAL4 and UAS-DREFIR resulted in a split thorax phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed the reduction in DREF knockdown clones. These findings indicate that DREF is involved in regulation of Drosophila thorax development via actions on the JNK pathway.

XNP/dATRX interacts with DREF in the chromatin to regulate gene expression.

The ATRX gene encodes a chromatin remodeling protein that has two important domains, a helicase/ATPase domain and a domain composed of two zinc fingers called the ADD domain. The ADD domain binds to histone tails and has been proposed to mediate their binding to chromatin. The putative ATRX homolog in Drosophila (XNP/dATRX) has a conserved helicase/ATPase domain but lacks the ADD domain. In this study, we propose that XNP/dATRX interacts with other proteins with chromatin-binding domains to recognize specific regions of chromatin to regulate gene expression. We report a novel functional interaction between XNP/dATRX and the cell proliferation factor DREF in the expression of pannier (pnr). DREF binds to DNA-replication elements (DRE) at the pnr promoter to modulate pnr expression. XNP/dATRX interacts with DREF, and the contact between the two factors occurs at the DRE sites, resulting in transcriptional repression of pnr. The occupancy of XNP/dATRX at the DRE, depends on DNA binding of DREF at this site. Interestingly, XNP/dATRX regulates some, but not all of the genes modulated by DREF, suggesting a promoter-specific role of XNP/dATRX in gene regulation. This work establishes that XNP/dATRX directly contacts the transcriptional activator DREF in the chromatin to regulate gene expression.

Trafficking and chromatin dynamics of holocarboxylase synthetase during development of Drosophila melanogaster.

This work examines the cellular localization of holocarboxylase synthetase (HCS) and its association to chromatin during different stages of development of Drosophila melanogaster. While HCS is well known for its role in the attachment of biotin to biotin-dependent carboxylase, it also regulates the transcription of HCS and carboxylases genes by triggering a cGMP-dependent signal transduction cascade. Further, its presence in the nucleus of cells suggests additional regulatory roles, but the mechanism involved has remained elusive. In this study, we show in D. melanogaster that HCS migrates to the nucleus at the gastrulation stage. In polytene chromosomes, it is associated to heterochromatin bands where it co-localizes with histone 3 trimethylated at lysine 9 (H3K9met3) but not with the euchromatin mark histone 3 acetylated at lysine 9 (H3K9ac). Further, we demonstrate the association of HCS with the hsp70 promoter by immunofluorescence and chromatin immuno-precipitation (ChIP) of associated DNA sequences. We demonstrate the occupancy of HCS to the core promoter region of the transcriptionally inactive hsp70 gene. On heat-shock activation of the hsp70 promoter, HCS is displaced and the promoter region becomes enriched with the TFIIH subunits XPD and XPB and elongating RNA pol II, the latter also demonstrated using ChIP assays. We suggest that HCS may have a role in the repression of gene expression through a mechanism involving its trafficking to the nucleus and interaction with heterochromatic sites coincident with H3K9met3.

Drosophila p53 is required to increase the levels of the dKDM4B demethylase after UV-induced DNA damage to demethylate histone H3 lysine 9.

Chromatin undergoes a variety of changes in response to UV-induced DNA damage, including histone acetylation. In human and Drosophila cells, this response is affected by mutations in the tumor suppressor p53. In this work, we report that there is a global decrease in trimethylated Lys-9 in histone H3 (H3K9me3) in salivary gland cells in wild type flies in response to UV irradiation. In contrast, flies with mutations in the Dmp53 gene have reduced basal levels of H3K9me3, which are then increased after UV irradiation. The reduction of H3K9me3 in response to DNA damage occurs preferentially in heterochromatin. Our experiments demonstrate that UV irradiation enhances the levels of Lys-9 demethylase (dKDM4B) transcript and protein in wild type flies, but not in Dmp53 mutant flies. Dmp53 binds to a DNA element in the dKdm4B gene as a response to UV irradiation. Furthermore, heterozygous mutants for the dKdm4B gene are more sensitive to UV irradiation; they are deficient in the removal of cyclobutane-pyrimidine dimers, and the decrease of H3K9me3 levels following DNA damage is not observed in dKdm4B mutant flies. We propose that in response to UV irradiation, Dmp53 enhances the expression of the dKDM4B histone demethylase, which demethylates H3K9me3 preferentially in heterochromatin regions. This mechanism appears to be essential for the proper function of the nucleotide excision repair system.

Globin genes transcriptional switching, chromatin structure and linked lessons to epigenetics in cancer: a comparative overview.

At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.

TFIIH trafficking and its nuclear assembly during early Drosophila embryo development.

We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.