Crosstalk Between CARM1 and ESRs
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Crosstalk Between CARM1 and ESRs

The ESRs (Estrogen Receptors) are ligand-dependent transcription factors and are important Nuclear Hormone Receptors that act as regulators of cell growth, differentiation and malignant transformation. Transcriptional activation by ESRs are accomplished through specific and general cofactor complexes that assemble with the receptor at target promoters to regulate transcription. The chief ligand for ESR is the ovarian steroid hormone Estrogen, which has a primary role in the establishment and maintenance of reproductive function (Ref.1). Naturally occurring forms of Estrogen are Estradiol, Estriol, and Estrone. Estradiol is the most commonly occurring form of Estrogen in non-pregnant women. Binding of Estrogen to the ESR promotes a conformational change in the receptor and the formation of a surface for protein-protein contacts between receptor domains and co-activators. The physiological effects of Estrogen are mediated by the intracellular ESR, which regulate transcription of target genes in form of homo- and hetrodimers, through binding to specific palindromic DNA target sequences known as EREs (Estrogen Response Elements) (Ref.2).

Like all NRs (Nuclear Receptors), the ESR contains at least two AFs (Transcription Activation Function Domains); constitutively active AF1 (Activation Function-1) in the N-terminus of the protein and ligand-dependent AF2 at the ESR C-terminus. These AFs represent surfaces capable of interaction with GTFs (General Transcription Factors) and additional transcriptional regulatory factors termed co-activators. Estrogen binding to the ESRs promotes the protein-protein contacts between AF2 and co-activators. Apart from AFs, the ESRs also contain the NLS (Nuclear Localization Signal) motifs and a central bipartite zinc finger DBD (DNA Binding Domain), which bind to the EREs in the DNA (Ref.3). Among the numerous co-activators of ESR the best characterized is the p160 family of proteins comprising of SRC1 (Steroid Receptor Coactivator-1)/NCOA1 (Nuclear Receptor Coactivator-1); GRIP1 (Glucocorticoid Receptor-Interacting Protein-1)/NCOA2 (Nuclear Receptor Coactivator-2); and RAC3 (Receptor-Associated Coactivator-3)/NCOA3(Nuclear Receptor Coactivator-3). The p160 proteins participate in transcriptional activation by ESR through AF2 and interact with the receptor in a ligand-dependent manner. The p160s also interact with ESR N-terminal region and increase AF1 transactivation independent of ligand (Ref.4).

These interactions are mediated by two distinct regions on the p160s; the central NR (Nuclear Receptor) Boxes bind to the ESR AF2 Domain, whereas the p160 C-terminus interacts with the ESR N-terminal A/B domain (also known as AF1). The p160s enhance ESR transactivation by recruiting other transcriptional regulatory factors through two Activation Domains, AD1 and AD2. AD1 interacts with CBP (CREB-Binding Protein) and p300, whereas AD2 associates with CARM1 (Coactivator-Associated Arginine Methyltransferase-1) and PRMT1 (Protein Arginine N-Methyltransferase-1) (Ref.5). ESR transcriptional activation is also increased by over-expression of RhoGDIs (Rho Guanine Nucleotide Dissociation Inhibitors), and this effect is mediated through an inhibition of RhoGTPases. The RhoGDI signal is transduced to ESR by CBP and p300 through p160s. RhoGDI-dependent increase in ESR transactivation is dependent on the ESR AF2 coactivator binding site. Indeed, RhoGDI cooperates with p160s to increase ESR ligand-independent and ligand-dependent transactivation and also enhances p160 transcriptional activity when p160s are tethered to DNA. The p160 activation domain, AD1, which binds CBP and p300, is necessary for RhoGDI to modulate p160 activity. The opposing effects of RhoGTPases and ESRs on cell invasion is consistent where RhoGTPases inhibit ESR transcriptional activity, thereby enhancing cell motility by blocking ESR target genes that suppress cell migration. In contrast, RhoGDI over-expression promotes expression of ESR target genes that restrain cell invasion by inhibiting RhoGTPases. Therefore the effect of the Rho Signaling Pathway on endogenous genes regulated by ESRs determines that RhoGDI specifically regulates genes involved in cell migration and invasion that correlate with breast tumor progression (Ref.4).

CBP, p300, CARM1 and PRMT1 are recruited to ESRs by p160s in response to Estrogen, where HAT (Histone Acetyltransferase) and HMT (Histone Methyltransferase) activity of these proteins stimulate transcription by remodeling Chromatin through subsequent Histone acetylation and methylation in the vicinity of ERE (Ref.6 & 7). Covalent post-translational modifications of Histone N-termini, such as acetylation, phosphorylation and methylation, play a fundamental role in chromatin structure and transcriptional regulation. Both PRMT1 and CARM1 co-activate ESR regulated gene expression and their co-activator potential is dependent on an intact methyltransferase domain, which aids in methyl group transfer along with S-AdoMet (S-Adenosylmethionine). CARM1 methylates Histone-H3 at Arg17 (Arginine 17) and PRMT1 specifically methylates Arg3 (Arginine 3) of Histone-H4 in the Nucleosome Complexes, respectively. The methylation of Arginines within Histones represents an activating step in mammalian gene transcription (Ref.8 & 9).

HMT activity by CARM1 plays a key role in ESR transcriptional regulation. The targets of CARM1, the core Histone-H3 and Histone-H4 are also, the targets of the HAT activity of CBP and p300 co-activators on Lys (Lysine) positions. HAT activities of p300, CBP and PCAF (p300/CBP-Associated Factor) are required for their co-activator function. They act synergistically with Acetyl-CoA (Acetyl-Coenzyme-A) and HAT1 (Histone Acetyltransferase-1) to enhance ESR function. The HAT activity is also responsive to DNA repair signals. Recruitment of CARM1 to the promoter region by binding to co-activators increases Histone methylation and makes promoter regions more accessible for transcription. Another target of CARM1 HMT activity is a co-activator it interacts with, CBP. Methylation of CBP by CARM1 blocks CBP from acting as a co-activator and redirects the limited CBP pool in the cell to be available for ESRs. Methylated CBP fails to mediate transcription because methylation blocks CBP interaction with other proteins (Ref.10 & 11). CARM1, PRMT1, CBP and p300 further stimulate ESR transactivation by interacting with components of the Basal Transcriptional Complex such as RNA Pol II (RNA Polymerase-II), TBP (TATA Box-Binding Protein), TFIIA (Transcription Factor-IIA), TFIIB, TFIIE, TFIIF, TFIIH (General Transcription Factor IIH Polypeptide) and TAFs (TBP Associated Factors), and facilitate an association with other transcription factors and co-regulators such as PCAF. TFIIs help to recruit RNA Pol II . CARM1 also acts as a co-activator for the myogenic transcription factor MEF2 (MADS Box Transcription Enhancer Factor-2 Polypeptide), and is necessary for normal muscle cell differentiation or Myogenesis. Thus a crosstalk between CARM1 and ESRs exists in accordance with Histone methylation and acetylation at various positions leading to hormonal response. A complex signal transduction by ESRs emerges that appears to rely on a collaboration of multiple factors for regulation of gene expression (Ref.4 & 12).

The covalent modifications of Histones are of central importance in the transcription regulation field. These are crucial towards differential gene expression involved in many cellular processes. Arginine methylation and Lysine acetylation of Histones give new insights into the regulation of ESR gene activity. They provide direct evidence that CARM1-mediated methylation of Histones takes place during transcriptional activation in vivo (Ref.13). In addition, the ability of these co-activators to acetylate and make contact with other components of the transcription machinery also helps to transmit the activating signal from the transcriptional activator/co-activator complex to the transcription machinery. CARM1 has both chromatin and non-chromatin substrates and methylation process acts as a unique transcriptional switch. The methylation of CBP selectively impairs transcription while stimulating ESR target genes. As CBP and p300 signaling pathways are developmentally and physiologically important, CARM1 methylation may have unexpectedly broad biological significance (Ref.14).