Hormones and AID: balancing immunity and autoimmunity.

The human immune system is a complex dynamic network of soluble factors and specialized cells that can and need to act in an instance or keep a lifelong protection, with the consequence that health has to be maintained through genetic and environmental stimuli. Autoimmunity is a multifactorial disease, where this combination of genetic predisposition and environmental factors lead to disease etiology. As some autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) or other B cell autoimmunities have a very strong female gender bias, hormones, especially estrogen, have been implicated as environmental factors in driving the disease. One of the key regulators of B cell development is activation-induced deaminase (AID), as its molecular mechanism of cytosine deamination induces immunoglobulin affinity maturation and antibody class switching. In this review we will highlight some of the recent findings of how estrogen directly and indirectly activates AID expression, which in turn can lead to immune hyper-stimulation. Those regulatory pathways can be direct when the estrogen receptor (ER) binds the AID promoter, or indirect via activation of transcription factors that enhance AID expression (e.g., HoxC4). Estrogen's influence on AID will also be discussed in terms of microRNA processing for miRNA-155 and miRNA-181b. Important other external stimuli, such as EBV virus, in conjunction with estrogen can add another layer of regulation during autoimmune disease progression. Understanding these pathways will become more important as AID has now been implicated to play an important role in immune tolerance and actual elimination of autoantibodies.

5-Methylcytosine DNA demethylation: more than losing a methyl group.

Demethylation of 5-methylcytosine in DNA is integral to the maintenance of an intact epigenome. The balance between the presence or absence of 5-methylcytosine determines many physiological aspects of cell metabolism, with a turnover that can be measured in minutes to years. Biochemically, addition of the methyl group is shared among all living kingdoms and has been well characterized, whereas the removal or reversion of this mark seems diverse and much less understood. Here, we present a summary of how DNA demethylation can be initiated directly, utilizing the ten-eleven translocation (TET) family of proteins, activation-induced deaminase (AID), or other DNA modifying enzymes, or indirectly, via transcription, RNA metabolism, or DNA repair; how intermediates in those pathways are substrates of the DNA repair machinery; and how demethylation pathways are linked and possibly balanced, avoiding mutations.

A role for the RNA pol II-associated PAF complex in AID-induced immune diversification.

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

AID enzymatic activity is inversely proportional to the size of cytosine C5 orbital cloud.

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl >> hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal.

AIDing the immune system-DIAbolic in cancer.

Activation induced deaminase (AID) has evolved with the immune system to enhance the ability of antibodies to bind and eliminate pathogens. It is a member of the AID/APOBEC family of proteins, which deaminate cytosine (or 5-methyl cytosine) in DNA leading to uracil (thymidine). These base modifications can lead to repair, DNA demethylation, mutagenesis, recombination, or viral/foreign DNA elimination. Because of their physiological function, their ubiquitous expression, and hormonal regulation (e.g. estrogen), these proteins play an important role in oncogenesis, with AID being directly implicated in B cell lymphomas. The targeting preference of each DNA deaminase provides a means to identify their mutation foot-print in tumours, and have implicated them in mutating the genome, including the loci of tumour suppressors, of various cancers (e.g. breast). In this special issue devoted to understanding AID function and regulation, leading members of the field discuss all aspects from AID transcriptional regulation, mRNA turnover, protein expression, modification, and transport, to complex formation, targeting and enzymatic turnover. AID's function will be discussed in context of DNA repair and how changes of key components of each pathway have an influence on the overall efficacy of targeted DNA deamination.

Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes.

Noncoding RNAs are important components of regulatory networks controlling the epigenetic state of chromatin. We analyzed the role of pRNA (promoter-associated RNA), a noncoding RNA that is complementary to the rDNA promoter, in mediating de novo CpG methylation of rRNA genes (rDNA). We show that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling new mechanism of RNA-dependent DNA methylation, suggesting that recruitment of DNMT3b by DNA:RNA triplexes may be a common and generally used pathway in epigenetic regulation.

Intergenic transcripts originating from a subclass of ribosomal DNA repeats silence ribosomal RNA genes in trans.

Epigenetic silencing of a fraction of ribosomal DNA (rDNA) requires association of the nucleolar chromatin-remodelling complex NoRC to 150-250 nucleotide RNAs (pRNA) that originate from an RNA polymerase I promoter located in the intergenic spacer separating rDNA repeats. Here, we show that NoRC-associated pRNA is transcribed from a sub-fraction of hypomethylated rRNA genes during mid S phase, acting in trans to inherit DNA methylation and transcriptional repression of late-replicating silent rDNA copies. The results reveal variability between individual rDNA clusters with distinct functional consequences.

Reversible acetylation of the chromatin remodelling complex NoRC is required for non-coding RNA-dependent silencing.

The SNF2h (sucrose non-fermenting protein 2 homologue)-containing chromatin-remodelling complex NoRC silences a fraction of ribosomal RNA genes (rDNA) by establishing a heterochromatic structure at the rDNA promoter. Here we show that the acetyltransferase MOF (males absent on the first) acetylates TIP5, the largest subunit of NoRC, at a single lysine residue, K633, adjacent to the TIP5 RNA-binding domain, and that the NAD(+)-dependent deacetylase SIRT1 (sirtuin-1) removes the acetyl group from K633. Acetylation regulates the interaction of NoRC with promoter-associated RNA (pRNA), which in turn affects heterochromatin formation, nucleosome positioning and rDNA silencing. Significantly, NoRC acetylation is responsive to the intracellular energy status and fluctuates during S phase. Activation of SIRT1 on glucose deprivation leads to deacetylation of K633, enhanced pRNA binding and an increase in heterochromatic histone marks. These results suggest a mechanism that links the epigenetic state of rDNA to cell metabolism and reveal another layer of epigenetic control that involves post-translational modification of a chromatin remodelling complex.

TAF12 recruits Gadd45a and the nucleotide excision repair complex to the promoter of rRNA genes leading to active DNA demethylation.

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Intergenic transcripts regulate the epigenetic state of rRNA genes.

Transcripts originating from the intergenic spacer (IGS) that separates rRNA genes (rDNA) have been known for two decades; their biological role, however, is largely unknown. Here we show that IGS transcripts are required for establishing and maintaining a specific heterochromatic configuration at the promoter of a subset of rDNA arrays. The mechanism of action appears to be mediated through the interaction of TIP5, the large subunit of the chromatin remodeling complex NoRC, with 150-300 nucleotide RNAs that are complementary in sequence to the rDNA promoter. Mutations that abrogate RNA binding of TIP5 impair the association of NoRC with rDNA and fail to promote H3K9&H4K20 methylation and HP1 recruitment. Knockdown of IGS transcripts abolishes the nucleolar localization of NoRC, decreases DNA methylation, and enhances rDNA transcription. The results reveal an important contribution of processed IGS transcripts in chromatin structure and epigenetic control of the rDNA locus.

GvpE- and GvpD-mediated transcription regulation of the p-gvp genes encoding gas vesicles in Halobacterium salinarum.

The transcription of the 14 p-gvp genes involved in gas vesicle formation of Halobacterium salinarum PHH1 is driven by the four promoters pA, pD, pF and pO. The regulation of these promoters was investigated in Haloferax volcanii transformants with respect to the endogenous regulatory proteins GvpE and GvpD. Northern analyses demonstrated that the transcription derived from the pA and pD promoters was enhanced by GvpE, whereas the activities of the pF and pO promoters were not affected. Similar results were obtained using promoter fusions with the bgaH reporter gene encoding an enzyme with beta-galactosidase activity. The largest amount of specific beta-galactosidase activity was determined for pA-bgaH transformants, followed by pF-bgaH and pD-bgaH transformants. The presence of GvpE resulted in a severalfold induction of the pA and pD promoter, whereas the pF promoter was not affected. A lower GvpE-induced pA promoter activity was seen in the presence of GvpD in the pA-bgaH/DE(ex) transformants, suggesting a function of GvpD in repression. To determine the DNA sequences involved in the GvpE-mediated activation, a 50-nucleotide region of the pA promoter was investigated by 4-nucleotide scanning mutagenesis. Some of these mutations affected the basal transcription, especially mutations in the region of the TATA box and the putative BRE sequence element, and also around position -10. Mutant E, harbouring a sequence with greater identity to the consensus BRE element, showed a significantly enhanced basal promoter activity compared to wild-type. Mutations not affecting basal transcription, but yielding a reduced GvpE-mediated activation, were located immediately upstream of BRE. These results suggested that the transcription activation by GvpE is in close contact with the core transcription machinery.