An astounding property of the nervous system is its cellular diversity. This diversity, which was initially realized by morphological and electrophysiological differences, is ultimately produced by variations in gene-expression programs. In most cases, these variations are determined by external cues. However, a growing number of neuronal types have been identified in which inductive signals cannot explain the few but decisive transcriptional differences that cause cell diversification. Here, we show that heterochromatic silencing, which we find is governed by histone methyltransferases G9a (KMT1C) and GLP (KMT1D), is essential for stochastic and singular olfactory receptor (OR) expression. Deletion of G9a and GLP dramatically reduces the complexity of the OR transcriptome, resulting in transcriptional domination by a few ORs and loss of singularity in OR expression. Thus, our data suggest that, in addition to its previously known functions, heterochromatin creates an epigenetic platform that affords stochastic, mutually exclusive gene choices and promotes cellular diversity.

The olfactory receptor (OR) genes are the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion in olfactory neurons. Using a high-throughput approach, we mapped the transcription start sites of 1085 of the 1400 murine OR genes and performed computational analysis that revealed potential transcription factor binding sites shared by the majority of these promoters. Our analysis produced a hierarchical model for OR promoter recognition in which unusually high AT content, a unique epigenetic signature, and a stereotypically positioned O/E site distinguish OR promoters from the rest of the murine promoters. Our computations revealed an intriguing correlation between promoter AT content and evolutionary plasticity, as the most AT-rich promoters regulate rapidly evolving gene families. Within the AT-rich promoter category the position of the TATA-box does not correlate with the transcription start site. Instead, a spike in GC composition might define the exact location of the TSS, introducing the concept of “genomic contrast” in transcriptional regulation. Finally, our experiments show that genomic neighborhood rather than promoter sequence correlates with the probability of different OR genes to be expressed in the same olfactory cell.

Human kallikrein 5 (KLK5) is a member of the human kallikrein gene family of serine proteases. Preliminary results indicate that the protein, hK5, may be a potential serological marker for breast and ovarian cancer. Other studies implicate hK5 with skin desquamation and skin diseases. To gain further insights on hK5 physiological functions, we studied its substrate specificity, the regulation of its activity by various inhibitors, and identified candidate physiological substrates. After producing and purifying recombinant hK5 in yeast, we determined the k_cat/K_m ratio of the fluorogenic substrates Gly-Pro-Arg-AMC and Gly-Pro-Lys-AMC, and showed that it has trypsin-like activity with strong preference for Arg over Lys in the P1 position. The serpins α₂-antiplasmin and antithrombin were able to inhibit hK5 with an inhibition constant (k₊₂/K_i) of 1.0 × 10^{– 2}and 4.2 × 10^–4 m^–1 min^–1, respectively. No inhibition was observed with the serpins α₁-antitrypsin and α₁-antichymotrypsin, although α₂-macroglobulin partially inhibited hK5 at high concentrations. We also demonstrated that hK5 can efficiently digest the extracellular matrix components, collagens type I, II, III, and IV, fibronectin, and laminin. Furthermore, our results suggest that hK5 can potentially release (a) angiostatin 4.5 from plasminogen, (b) "cystatin-like domain 3" from low molecular weight kininogen, and (c) fibrinopeptide B and peptide β15-42 from the Bβ chain of fibrinogen. hK5 could also play a role in the regulation of the binding of plasminogen activator inhibitor 1 to vitronectin. Our findings suggest that hK5 may be implicated in tumor progression, particularly in invasion and angiogenesis, and may represent a novel therapeutic target.