Regulation of Primary Response Genes Trent Fowler, Ranjan Sen, Ananda L. Roy  Molecular Cell  Volume 44, Issue 3, Pages 348-360 (November 2011) DOI: 10.1016/j.molcel.2011.09.014 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Biological Outcome in Response to Transient versus Sustained Signaling Resting cells can be triggered either by transient signaling or by sustained signaling, leading to different biological outcomes. Transient signaling might elicit the cellular initiation program, while sustained signaling might elicit the maintenance program. In addition, a different set of genes is regulated (by both induction and repression) upon transient (Gene Set “T”) versus sustained signaling (Gene Set “S”) with an overlapping set (Common Genes). Molecular Cell 2011 44, 348-360DOI: (10.1016/j.molcel.2011.09.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Transcriptional Regulation of PRGs by SRF and Cofactors SRF regulates PRGs by virtue of association with different cofactors. In the absence of signaling (serum starvation), MAL can associate with G-actin, which prevents its nuclear residency as well as transcriptional activity. Upon signaling with serum via the Rho pathway, MAL dissociates from actin, which enhances its nuclear import and transcriptional activity, resulting in its recruitment to target genes. Concomitantly, actin polymerization is enhanced under these conditions. These changes in cytoskeletal organization are associated with proliferation, metastasis. In cardiac muscle cells, SRF can associate with other MRTF family cofactors (e.g., Myocardin) and regulate cardiac responses. In T cells, SRF associates with the NURF chromatin remodeling complex and regulates thymocyte development. In neuronal cells, SRF and its cofactors or CREB regulate different sets of genes to regulate neuronal plasticity versus survival, respectively. Note that not all cofactors associate with SRF at the same time in the same cells. For the sake of simplicity, only the MRTF/MKL cofactors are shown. The basal transcriptional machinery, comprised of general transcription factors, is indicated by the light blue oval. Molecular Cell 2011 44, 348-360DOI: (10.1016/j.molcel.2011.09.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Direct DNA Binding by ERK to Regulate PRGs (A) In resting state, ERK2 directly binds to DNA, preventing binding of C/EBP-β. This results in transcriptional repression of PRGs. (B) Upon transient IFN-γ signaling, ERK2 is released from the DNA and an overlapping site is occupied by C/EBP-β, resulting in transcriptional activation of target genes. (C) Upon sustained IFN-γ signaling, ERK2 DNA binding reappears resulting in repression. It is unknown at present how ERK2 represses transcription. In T cells, upon cessation of signaling, unphosphorylated RNA Pol II and the histone acetyl transferase, p300, remain associated with PRGs, thereby “bookmarking” these genes and allowing them to be reactivated by nonmitogenic stimulation alone. Please note, the figure represents two different observations from two distinct cell types, and thus ERK2 target genes may not be bookmarked. The blue oval represents the basal machinery. Elongating RNA Pol II is phosphorylated at S-2 and S-5. Molecular Cell 2011 44, 348-360DOI: (10.1016/j.molcel.2011.09.014) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Integrated Model for Transcriptional Regulation of PRGs (A) In the basal state, the PRGs exhibit a constitutive level of HeK4me3 and H3Ac (data not shown for the sake of simplicity) and are bound by Sp1 and SRF. Transcriptional corepressors NCo-R and HDACs are also recruited to lower the basal expression. Both pausing of RNA Pol II and production of unstable primary transcripts have been shown. Paused RNA Pol II, bearing S-5-phosphorylated CTD, is associated with DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). MiRNAs, e.g., miR-155, miR-191, and miR-499, can negatively regulate expression of PRGs, thereby suppressing growth or cardiac stress. (B) Upon signaling, both H3K4me3 and H3Ac modifications increase, as do H3S01P and H4K16Ac. Phosphorylation of H3S10 is mediated by PIM1 kinase. Phosphorylated H3S10 recruits 14-3-3, which further recruits the histone acetyl transferase, MOF. MOF mediates H4K16 acetylation that in turn enhances BRD4 association and P-TEFb recruitment. NF-κB, NFI, and phosphorylated ELK1 (P-ELK1) are additionally recruited to these genes. The Mediator subcomplex MED23 or CDK8 is also associated with the promoters upon signaling. The curved arrows indicate that both NF-κB and CDK8 further enhance recruitment of BRD4 and P-TEFb. P-TEFb recruitment enhances phosphorylation of RNA Pol II S-2 as well as NELF and DSIF, resulting in release of phosphorylated NELF (P-NELF). Phosphorylated DSIF possibly remains associated with elongating RNA Pol II. In addition, the splicing factor U2AF65 (along with factors like Prp-19) is also recruited to PRGs, which directly interacts with activated RNA Pol II, thereby connecting splicing to transcription. Under these conditions (induced state), mature and stable transcripts are produced. Upon signaling, SRF can also trigger ripple effects on neighboring genes, which are not normally dependent on SRF, rendering them active. Furthermore, upon signaling, enhancer RNAs (eRNAs, pink rectangle) are produced, which communicate (indicated by a curved arrow) with the basal machinery (indicated by the light blue oval) to enhance transcription. It is important to note that not all observations depicted here are demonstrated in multiple cell types and/or under different signaling conditions. Molecular Cell 2011 44, 348-360DOI: (10.1016/j.molcel.2011.09.014) Copyright © 2011 Elsevier Inc. Terms and Conditions