A team led by researchers at Princeton University has built a global protein interaction network for all 11 human histone deacetylases.
The project, representing the most comprehensive characterization to date of the HDAC interactome, identified more than 200 previously unreported interactions, and offer "an important resource for elucidating HDAC functions in health and disease," Princeton researcher Ileana Cristea, leader of the effort, told ProteoMonitor.
In the study, detailed in a paper published last week in Molecular Systems Biology, the researchers combined immunoaffinity purification with label-free and metabolic-labeling mass spec techniques to both identify HDAC interactions and establish these interactions' relative stability.
Among the group's novel findings was establishing HDAC11 as a member of the survival of motor neuron complex with a role in mRNA splicing. Also of interest, Cristea said, was the finding of interactions between HDAC1 and several enzymes involved in histone demethylation as well as the interaction of HDAC8 with members of the cohesion complex.
Key regulators of gene transcription, HDAC dysfunction has been linked to a variety of diseases including cancer, infection, and heart disease. Given this, these proteins are attractive drug targets. However, Cristea noted, due to their involvement in a wide range of cellular processes, treatments targeting them can be highly cytotoxic – making better understandings of their interactions key to drug development.
"Even when a small molecule is specific enough to inhibit [only] one HDAC, it will still affect the functions of numerous HDAC-containing complexes and diverse cellular pathways," she said. "So, there is a need to better define the distinct HDAC complexes."
"In the long run," she noted, "these sub-complexes can become more specific targets for therapeutic intervention."
To build their interaction network, Cristea and her colleagues combined immunoaffinity pull-down of the interacting complexes with mass spec analysis on a Thermo Fisher Scientific LTQ-Orbitrap Velos. They assessed the specificity of the observed interactions using the SAINT – Significance Analysis of INTeractions – algorithm.
Via this analysis, they identified 281 proteins as potential specific interactors across all 11 HDACs with which they constructed an HDAC interaction network profiling the associations between HDACs and the various prey proteins.
Using metabolic labeling, the researchers also measured the stability of the observed HDAC-protein interactions. Following the I-DIRT -- isotopic differentiation of interactions as random or targeted – protocol first published by Rockefeller University researchers in 2005, the Princeton team mixed SILAC-labeled wild-type T cells with T cells expressing the target HDAC protein expressed in SILAC "light" media. Then, by quantifying the SILAC peptide pairs and measuring the isotope ratios they were able to determine the relative stability of the various interactions, with transient interactions having "light"-"heavy" ratios approaching 0.50 and more stable interactions having ratios approaching 1.0.
The addition of the stability data added a level of analysis complementary to the label-free SAINT-based work, Cristea noted.
For instance, "proteins that are identified [as potential interactors] with a low number of spectra may not reach the requirements to pass the SAINT threshold for specificity," she said. But "I-DIRT may still identify these as specific interactions by measuring the ratios of heavy and light signals for the few identified peptides."
At the same time, she added, the I-DIRT approach would tend to miss as non-specific fast-occurring interactions. Provided these proteins had sufficient spectral counts, though, they would be picked up by the SAINT-based method.
"So integrating these two approaches has real benefits," Cristea said.
One interesting observation to emerge from the stability data was that the less stable interactions tended to have roles related to transcription. This finding, Cristea suggested, could offer insight into how HDACs target various transcription factors.
A key question the researchers had, she said, was "are HDACs binding to transcription factors and then forming specialized complexes or are they already present within distinct complexes prior to their binding to transcription factors?"
"Our results suggest that HDACs are part of specialized and pre-assembled complexes that then interact with transcription factors," Cristea said, adding that it "is tempting to hypothesize that these pre-assembled functional deacetylase units could allow for more rapid alterations to chromatin structures."
In addition to identifying a number of novel interactors, the researchers identified all the known major components of several key HDAC1 and HDAC2 complexes, Cristea said, adding that this was an important validation of their approach. She noted, however, that given the stringency of the requirements they used for identifying interactions as specific, it is likely that a number of more transient HDAC interactions are not represented in their network.
"For example," she said, "we know from previous work in my lab that the class IIa HDACs HDAC4, HDAC5 and HDAC9 interact with and are phosphorylated by the kinase Aurora B. This interaction was not depicted in our stringently-filtered list of interactions."
Cristea noted as well that HDAC interactions are likely to be cell-type specific to an extent. "Therefore, more studies will be needed to characterize HDAC interactions in other biologically relevant cell systems," she said.
The MSB work was performed in T cells, a choice, Cristea said, the researchers made because of the critical role of HDACs in T cell biology. She cited as an example the use of small molecule HDAC inhibitors for treatment of cutaneous T cell lymphomas.
Beyond her broad study of HDAC interactions, Cristea is particularly interested in the roles of chromatin remodeling enzymes during viral infection. In fact, she said, her lab's interest in HDAC interactions "started from this perspective, when our studies of virus-host interactions showed that both DNA and RNA viruses target deacetylases during the progression of viral infections."
In light of this finding, "establishing this baseline of knowledge regarding the landscape of HDAC interactions is critical," Cristea said, noting that moving forward it would allow her lab "to properly investigate the infection-dependent changes in HDAC interactions and functions."