Biology subjects

Burkholder, N. T.

Publications and source records attributed to Burkholder, N. T..

3 recordsLinked to original sources

bioRxiv2026

Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein implicated in transcriptional control and oncogenesis, yet how its tandem bromodomains (BD1-2) contribute to nucleosomal engagement remains unresolved. Here we show that the tandem bromodomains of BRD4 cooperatively engage poly-acetylated histone H4 tails and nucleosomes in vitro and promote chromatin association in human cells. In stringent peptide pull-down and nucleosome-based biolayer interferometry assays, isolated BRD4 bromodomains bind weakly to poly-acetylated histone peptides and nucleosomes, whereas the tandem BD1-2 module binds much more robustly. These results closely mirror our observations in mammalian cells, where truncations lacking either bromodomain or pocket-disrupting mutations in either domain reduced chromatin association, with dual pocket disruption causing the strongest defect. In the BRD4 short isoform (BRD4-S), maximal chromatin association additionally required the region C-terminal to the BD2, which contains the basic residue-enriched interaction domain (BID) and extraterminal domain (ET), consistent with a multivalent chromatin engagement mechanism beyond the bromodomains alone. Functionally, dual pocket disruption attenuated BRD4-S-dependent breast cancer phenotypes, including impaired growth and reduced transwell migration. Together, these findings define how tandem bromodomains and adjacent BRD4-S regions cooperate to stabilize chromatin residence and inform therapeutic strategies aimed at more precisely disrupting BRD4 function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/719657v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@12d03beorg.highwire.dtl.DTLVardef@50cc45org.highwire.dtl.DTLVardef@92e3ecorg.highwire.dtl.DTLVardef@1b1a26d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Using structurally fungible biosensors to evolve improved alkaloid biosyntheses

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can greatly improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a unique combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors, and using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (EC50 <30 M) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures reveal multiple evolutionary avenues for the fungible effector binding site, and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, an immediate precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors now enable the rapid engineering of pathways for therapeutic alkaloids.

synthetic biology↗

bioRxiv2019

Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase IIs C-terminal domain (CTD) and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes a reduction of phosphorylated Ser2 and accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTDs coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

biochemistry↗