Chromatin structural dynamics regulate diverse cellular functions that influence survival, growth, and proliferation, and disruption of chromatin homeostasis is thought to fundamentally impact on the development and progression of cancers and other diseases. One of the major mechanisms for regulating chromatin structure involves the reversible covalent post-translational modification of histone proteins by chemical moieties such as acetyl-, methyl- and phospho- groups.  These chemical marks constitute an epigenetic code that can be maintained in dividing cells, and inherited across generations. Further, epigenetic alterations at chromatin in disease are potentially reversible, and thus, strategies aimed at negating these changes hold great promise as therapeutic targets.  Our work focuses on understanding at the basic molecular level how chromatin modifications are sensed and transduced to effect biological outcomes such as DNA damage responses, and how disruption in these mechanisms can lead to pathologic states.

General areas of chromatin research we investigate include:

(1)The identification of novel chromatin effector domains.
(2)Signaling role of chromatin modifications in DNA damage response pathways.
(3)Chromatin regulatory functions and molecular targets of nuclear PtdInsPs.

(1) Identification of the PHD finger as a novel recognition module for histone H3 trimethylated at lysine 4.

Different histone modifications are linked to discrete chromatin states and are thought to regulate the extent of accessibility of DNA to transacting factors.  One of the marks most clearly associated with euchromatic genomic regions is methylation at lysine 4 of histone H3  (H3K4).  H3K4 can be mono-, di- or tri-methylated, with the trimethylated species (H3K4me3) preferentially detected at active genes.  Lysine methylation does not neutralize the cationic charge of the e-NH2, but does change the hydrophobicity and upon higher methylation states, the steric bulk of the lysine side chain.  Thus, addition of methyl moieties to lysines is believed to not intrinsically affect chromatin structure, and rather methylation is proposed to create a distinct molecular architecture that is specifically recognized by specialized binding domains present within chromatin-regulatory proteins.  In this context, the proteins and domains that recognize this modification may define the functional consequences of histone methylation.

We have identified a novel class of H3K4me3 effector domains, the PHD fingers (Plant Homeodomain) from the ING (INhibitor of Growth) family of candidate tumor suppressor proteins (Shi et. al., 2006, Nature). The PHD finger is a signature chromatin-associated protein motif and is present throughout eukaryotic proteomes. Mutations in the PHD domains of many proteins are associated with cancers, immunodeficiency and mental retardation syndromes, and other genetic disorders. We have found that the PHD fingers present on the candidate tumor suppressor protein ING2 is a highly robust and specific binding module of H3K4me3. ING2 is a native subunit of a repressive mSin3a/HDAC1 histone deacetylase complex. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD finger stabilizes the mSin3a/HDAC1 complex at the promoters of proliferation genes, and suggests a novel mechanism by which H3K4me3 functions in active gene repression.

We are pursuing the hypothesis that by focusing HDAC1 repressor complexes on actively transcribed genes, recognition of H3K4me3 by ING2 may be important for the efficiency of acute gene repression. Such a mechanism may be particularly important in the context of cellular responses to acute stress, such as DNA damage, in which rapid shut-off of proliferation genes is critical to prevent propagation of cells harboring damaged DNA. We are also collaborating with the lab of Dr. Tanya Kutataladze at the University of Colorado Health Science Center to develop a better biophysical understanding of ING2 PHD finger recognition of H3K4me3. The crystal structure of the ING2 PHD finger complexed with H3K4me3 peptides at 2.0 Å resolution has been solved (shown below) and reveals the molecular basis for the high specificity of ING2 for trimethylated H3K4 versus other lysine residues (Pena et. al., 2006, Nature).

In addition to ING2, we have found that all the S. cerevisiae and Human INGs also function as robust and specific binding modules of H3K4me3 and H3K4me2. Because members of the ING family are native subunits of different histone acetyl-transferase (HAT) and deacetylase (HDAC) enzyme complexes, binding of the PHD domains of the other ING proteins may link H3K4 methylation to additional cellular functions.

In addition to the ING PHD fingers, we have evidence that other PHD fingers present on diverse nuclear proteins also function as methyl-lysine recognition modules, suggesting a more general role for PHD domains as methyl-lysine effector domains. One aim of our research will be to characterize and elucidate the physiologic functions of these interactions.

(2) Signaling role of chromatin modifications in DNA damage response pathways.

The human genome is continually exposed to environmental and metabolic agents that can trigger DNA damage, the accumulation of which is strongly linked to tumorigenesis and the pathogenesis of many other diseases. Interruption of both stands of DNA (double-strand break (DSB)), is a particularly dangerous form of DNA damage as the free ends of the broken DNA can randomly integrate into the genome to cause aberrant chromosomal rearrangements and genomic instability. There is a great deal of evidence that DSB formation triggers alterations in chromatin structure, including dynamic and specific post-translational covalent modifications of histone proteins. It is hypothesized that the resulting pattern of histone modifications establishes a distinct molecular architecture that is recognized by and leads to recruitment of different DNA damage effector activities. In this way, chromatin modifications are thought to play critical roles in the surveillance, detection and repair of DSBs. We would like to develop systems to study these changes in mammalian cells with the aim of gaining new insight into the molecular mechanism of how a DNA damage epigenetic program is established.

(3) Chromatin regulatory functions and molecular targets of nuclear PtdInsPs.

Phosphoinositides (PtdInsPs) are important lipid signaling molecules with well-established roles in regulating apoptosis, chemotaxis, vesicle trafficking, and other cellular functions.  Highlighting the physiologic importance of PtdInsP activity, dysregulation of PtdInsP homeostasis is implicated in tumorigenesis and other disease processes.  Most studies on PtdInsP regulatory mechanisms have centered on cytoplasmic processes and potential roles for PtdInsPs in the nucleus have been little explored.  Nevertheless, PtdInsPs and many of the enzymes that regulate their levels are found in the nucleus.  Moreover, levels of nuclear PtdInsPs undergo dynamic changes in response to specific stimuli, independent of cytoplasmic PtdInsP pools.  Indeed, there is evidence that PtdInsPs modulate a number of nuclear processes, but the molecular basis of these effects is poorly understood.

We have found that the PHD finger of ING2 functions as a dual-specificity module that binds independently to H3K4me3 and to the rare signaling PtdInsP, PtdIns(5)P (Gozani et. al., 2003, Cell). We are exploring the relationship between these two ligands, and postulate that PtdIns(5)P might regulate subnuclear trafficking of ING2 via interactions with the PHD finger, and thereby regulate chromatin biology.

Interestingly, a number of clinically important PtdInsP kinases and phosphatases, including PI3K, PTEN and SHIP2, translocate into the nucleus, often upon activation. The presence of these enzymes in the nucleus suggests that they regulate the nuclear levels of distinct PtdInsP species, and modulate as yet unknown nuclear processes.