The elongation stage of transcription is highly regulated in metazoans. We previously purified the AFF1-and AFF4-containing super elongation complex (SEC) as a major regulator of development and cancer pathogenesis. Here, we report the biochemical isolation of SEC-like 2 (SEC-L2) and SEC-like 3 (SEC-L3) containing AFF2 and AFF3 in association with P-TEFb, ENL/MLLT1, and AF9/MLLT3. The SEC family members demonstrate high levels of polymerase II (Pol II) C-terminal domain kinase activity; however, only SEC is required for the proper induction of the HSP70 gene upon stress. Genome-wide mRNA-Seq analyses demonstrated that SEC-L2 and SEC-L3 control the expression of different subsets of genes, while AFF4/SEC plays a more dominant role in rapid transcriptional induction in cells. MYC is one of the direct targets of AFF4/SEC, and SEC recruitment to the MYC gene regulates its expression in different cancer cells, including those in acute myeloid or lymphoid leukemia. These findings suggest that AFF4/SEC could be a potential therapeutic target for the treatment of leukemia or other cancers associated with MYC overexpression.T ranscription by RNA polymerase II (Pol II) is a finely tuned and multistep process (40,42,51). After the synthesis of the first few phosphodiester bonds, RNA Pol II escapes from the promoter and enters the productive elongation stage of transcription, depending on the presence of proper environmental signals (40). For decades, the preinitiation complex (PIC) assembly was thought to be the main target of regulation during the entire transcription process. Recently, however, a large number of studies have demonstrated that in addition to the regulation of PIC, promoter-proximal pausing by Pol II and its controlled release is a major regulatory step, especially on developmentally regulated genes (3,7,27,35,38,42,43,57). Multiple elongation factors regulating the elongation stage of transcription have been identified. These include P-TEFb (positive transcription elongation factor), DSIF (DRB sensitivity-inducing factor), NELF (negative transcription elongation factor), and ELL (eleven-nineteen lysinerich leukemia gene) (21,37,40,42,43). DSIF and NELF coordinately participate in setting up paused Pol II at the promoterproximal region (50, 53). The cyclin-dependent kinase 9 (CDK9) module of the P-TEFb complex phosphorylates serine 2 of the Pol II C-terminal domain (CTD), the SPT5 subunit of DSIF, and the E subunit of NELF, leading to the dissociation of paused Pol II from DSIF and NELF for productive elongation (12,21,29,37).The kinase activity of P-TEFb is tightly regulated in vivo through the formation of different complexes to achieve its regulation of transcription elongation. The inactive form of the P-TEFb complex contains 7SK-RNA, MEPCE, LARP7, and HEXIM1, which sequester PTEFb and inhibit its kinase activity (4, 18). The vast majority of P-TEFb exists in this inactive pool (36, 55). P-TEFb was later found to form a complex with the bromodomain protein BRD4. The BRD4/ P-TEFb complex, which can phos...

SUMMARY Eleven-nineteen Lysine-rich Leukemia (ELL) participates in the Super Elongation Complex (SEC) with the Pol II CTD kinase P-TEFb. SEC is a key regulator in the expression of HOX genes in Mixed Lineage Leukemia (MLL) -based hematological malignancies, in the control of induced gene expression early in development, and in immediate early gene transcription. Here, we identify an SEC-like complex in Drosophila, as well as a distinct ELL-containing complex that lacks P-TEFb and other components of SEC named the “little elongation complex” (LEC). LEC subunits are highly enriched at RNA Polymerase II (Pol II) -transcribed small nuclear RNA (snRNA) genes, and the loss of LEC results in decreased snRNA expression in both flies and mammals. The specialization of the SEC and LEC complexes for mRNA and snRNA-containing genes, respectively, suggests the presence of specific classes of elongation factors for each class of genes transcribed by RNA polymerase II.

SUMMARY The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA Polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the Little Elongation Complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL.

High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM ( n uclear ext ract p rotein- b inding m icroarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.

No abstract

Determining how DNA variants affect the binding of regulatory complexes to cisregulatory elements (CREs) and non-coding single-nucleotide polymorphisms (ncSNPs) is a challenge in genomics. To address this challenge, we have developed CASCADE (Comprehensive ASsessment of Complex Assembly at DNA Elements), which is a proteinbinding microarray (PBM)-based approach that allows for the high-throughput profiling of cofactor (COF) recruitment to DNA sequence variants. The method also enables one to infer the identity of the transcription factor-cofactor (TF-COF) complexes involved in COF recruitment.We use CASCADE to characterize regulatory complexes binding to CREs and SNP quantitative trait loci (SNP-QTLs) in resting and stimulated human macrophages. By profiling the recruitment of the acetyltransferase p300 and MLL methyltransferase component RBBP5, we identify key regulators of the chemokine CXCL10, and by profiling a set of five functionally diverse COFs we identify a prevalence of ETS sites mediating COF recruitment at SNP-QTLs in macrophages.Our results demonstrate that CASCADE is a customizable, high-throughput platform to link DNA variants with the biophysical complexes that mediate functions such as chromatin modification or remodeling in a cell state-specific manner. MainDetermining the impact of genetic variation on cis-regulatory elements (CREs), such as enhancers and promoters that control gene expression, remains a challenge in modern genomics. Genome-wide association studies (GWAS) have identified thousands of singlenucleotide polymorphisms (SNPs) associated with human diseases, but the causal variants and their biological effects remain largely unknown 1-3 . Variants underlying disease risk often function by altering CRE function and gene expression. For example, >50% of causal SNPs for autoimmune diseases are non-coding SNPs (ncSNPs) mapping to immune gene enhancers 4 . Therefore, a major challenge in understanding disease susceptibility is to determine how non-

Determining the biophysical principles that shape transcription factor (TF) binding in a cellspecific manner is key to quantitative models of gene expression. High-throughput (HT) in vitro methods measuring protein-DNA binding are invaluable for relating TF binding affinity to genome-wide binding; however, the impact of cell-specific post-translational modifications (PTMs) and cofactors are not routinely assessed. To address these limitations, we describe a new HT approach, called nextPBMs (nuclear extract protein-binding microarrays), to characterize TF binding that accounts for PTMs and endogenous cofactors. We use nextPBMs to examine the DNA binding of the lineage factor PU.1/Spi1 and IRF8 in human monocytes. We identify two binding modes for PU.1 in monocytes -autonomous binding unaffected by PTMs and cooperative binding with IRF8, and identify a single cooperative mode for IRF8. We characterize the DNA binding of PU.1:IRF8 complexes, and show how nextPBMs can be used to discover cell-specific cofactors and characterize TF cooperativity at single-nucleotide resolution. We show that chromatin state and cofactors both influence the affinity requirements for PU.1 binding sites. Furthermore, we find that the influences of cooperative (IRF8) and collaborative (C/EBPα) cofactors on PU.1-binding-site affinity are independent and additive.