The PAX3 paired domain and homeodomain function as a single binding module in vivo to regulate subnuclear localization and mobility by a mechanism that requires base-specific recognition.

The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.

Comparative analyses of SUV420H1 isoforms and SUV420H2 reveal differences in their cellular localization and effects on myogenic differentiation.

BACKGROUND: Methylation of histone H4 on lysine 20 plays critical roles in chromatin structure and function via mono- (H4K20me1), di- (H4K20me2), and trimethyl (H4K20me3) derivatives. In previous analyses of histone methylation dynamics in mid-gestation mouse embryos, we documented marked changes in H4K20 methylation during cell differentiation. These changes were particularly robust during myogenesis, both in vivo and in cell culture, where we observed a transition from H4K20me1 to H4K20me3. To assess the significance of this change, we used a gain-of-function strategy involving the lysine methyltransferases SUV420H1 and SUV420H2, which catalyze H4K20me2 and H4K20me3. At the same time, we characterized a second isoform of SUV420H1 (designated SUV420H1_i2) and compared the activity of all three SUV420H proteins with regard to localization and H4K20 methylation. PRINCIPAL FINDINGS: Immunofluorescence revealed that exogenous SUV420H1_i2 was distributed throughout the cell, while a substantial portion of SUV420H1_i1 and SUV420H2 displayed the expected association with constitutive heterochromatin. Moreover, SUV420H1_i2 distribution was unaffected by co-expression of heterochromatin protein-1α, which increased the targeting of SUV420H1_i1 and SUV420H2 to regions of pericentromeric heterochromatin. Consistent with their distributions, SUV420H1_i2 caused an increase in H4K20me3 levels throughout the nucleus, whereas SUV420H1_i1 and SUV420H2 facilitated an increase in pericentric H4K20me3. Striking differences continued when the SUV420H proteins were tested in the C2C12 myogenic model system. Specifically, although SUV420H1_i2 induced precocious appearance of the differentiation marker Myogenin in the presence of mitogens, only SUV420H2 maintained a Myogenin-enriched population over the course of differentiation. Paradoxically, SUV420H1_i1 could not be expressed in C2C12 cells, which suggests it is under post-transcriptional or post-translational control. CONCLUSIONS: These data indicate that SUV420H proteins differ substantially in their localization and activity. Importantly, SUV420H2 can induce a transition from H4K20me1 to H4K20me3 in regions of constitutive heterochromatin that is sufficient to enhance myogenic differentiation, suggesting it can act an as epigenetic 'switch' in this process.

Distinct dynamics and distribution of histone methyl-lysine derivatives in mouse development.

Histone methylation acts as an epigenetic regulator of chromatin activity through the modification of arginine and lysine residues on histones H3 and H4. In the case of lysine, this includes the formation of mono-, di-, or trimethyl groups, each of which is presumed to represent a distinct functional state at the cellular level. To examine the potential developmental roles of these modifications, we determined the global patterns of lysine methylation involving K9 on histone H3 and K20 on histone H4 in midgestation mouse embryos. For each lysine target site, we observed distinct subnuclear distributions of the mono- and trimethyl versions in 10T1/2 cells that were conserved within primary cultures and within the 3D-tissue architecture of the embryo. Interestingly, three of these modifications, histone H3 trimethyl K9, histone H4 monomethyl K20, and histone H4 trimethyl K20 exhibited marked differences in their distribution within the neuroepithelium. Specifically, both histone H3 trimethyl K9 and H4 monomethyl K20 were elevated in proliferating cells of the neural tube, which in the case of the K9 modification was limited to mitotic cells on the luminal surface. In contrast, histone H4 trimethyl K20 was progressively lost from these medial regions and became enriched in differentiating neurons in the ventrolateral neural tube. The inverse relationship of histone H4 K20 methyl derivatives is even more striking during skeletal and cardiac myogenesis where the accumulation of the trimethyl modification in pericentromeric heterochromatin suggests a role in gene silencing in postmitotic muscle cells. Importantly, our results establish that histone lysine methylation occurs in a highly dynamic manner that is consistent with their function in an epigenetic program for cell division and differentiation.