Structure of the super-elongation complex subunit AFF4 C-terminal homology domain reveals requirements for AFF homo- and heterodimerization

J Biol Chem. 2019 Jul 5;294(27):10663-10673. doi: 10.1074/jbc.RA119.008577. Epub 2019 May 30.


AF4/FMR2 family member 4 (AFF4) is the scaffold protein of the multisubunit super-elongation complex, which plays key roles in the release of RNA polymerase II from promoter-proximal pausing and in the transactivation of HIV-1 transcription. AFF4 consists of an intrinsically disordered N-terminal region that interacts with other super-elongation complex subunits and a C-terminal homology domain (CHD) that is conserved among AF4/FMR2 family proteins, including AFF1, AFF2, AFF3, and AFF4. Here, we solved the X-ray crystal structure of the CHD in human AFF4 (AFF4-CHD) to 2.2 Å resolution and characterized its biochemical properties. The structure disclosed that AFF4-CHD folds into a novel domain that consists of eight helices and is distantly related to tetratrico peptide repeat motifs. Our analyses further revealed that AFF4-CHD mediates the formation of an AFF4 homodimer or an AFF1-AFF4 heterodimer. Results from fluorescence anisotropy experiments suggested that AFF4-CHD interacts with both RNA and DNA in vitro Furthermore, we identified a surface loop region in AFF4-CHD as a substrate for the P-TEFb kinase cyclin-dependent kinase 9, which triggers release of polymerase II from promoter-proximal pausing sites. In conclusion, the AFF-CHD structure and biochemical analyses reported here reveal the molecular basis for the homo- and heterodimerization of AFF proteins and implicate the AFF4-CHD in nucleic acid interactions. The high conservation of the CHD among several other proteins suggests that our results are also relevant for understanding other CHD-containing proteins and their dimerization behavior.

Keywords: AF4/FMR2 family member 4 (AFF4); C-terminal homology domain (CHD); RNA polymerase II; X-ray crystallography; dimerization; gene regulation; phosphorylation; super elongation complex; transcription elongation factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Crystallography, X-Ray
  • DNA / chemistry
  • DNA / metabolism
  • Dimerization
  • Humans
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B / metabolism
  • Protein Folding
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • RNA / chemistry
  • RNA / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Sequence Alignment
  • Substrate Specificity
  • Transcriptional Elongation Factors / chemistry*
  • Transcriptional Elongation Factors / genetics
  • Transcriptional Elongation Factors / metabolism


  • AFF4 protein, human
  • Protein Subunits
  • Recombinant Proteins
  • Transcriptional Elongation Factors
  • RNA
  • DNA
  • Positive Transcriptional Elongation Factor B

Associated data

  • PDB/6GKG-F