Sleep loss has a profound, negative impact on cognition, learning, mood, and diverse aspects of mental health 
. Although chronic sleep debt is a growing problem in our society, the molecular and cellular sources of its deleterious effects remain poorly understood. Sleep is known to be regulated by the tight interaction between two main processes 
: a sleep homeostat that tracks sleep need according to the duration of time spent awake and asleep, and a circadian timing system that determines the propensity for wakefulness according to an about 24 h rhythm.
At the molecular level, the regulation of the circadian timing system has been extensively studied and it is now well-established that circadian rhythms originate from interacting transcriptional-translational feedback loops involving clock genes and their protein products 
. In contrast, the molecular wiring of the sleep homeostat remains to be defined. The function of the core clock transcription factors CLOCK, NPAS2, and BMAL1 resides in their hetero-dimerization and binding to E-box (CANNTG) or E-box like (E'-box) elements of target genes from which transcription is initiated 
. Some specific targets such as Period
)-1, and -2
, and Cryptochrome (Cry)-1,
genes subsequently provide negative feedback by interfering with the CLOCK::BMAL1 or NPAS2::BMAL1 transcriptional complexes thereby inhibiting their own transcription 
. In addition, other target genes such as the orphan nuclear receptor families Rev-Erb
, respectively suppress and promote the expression of Npas2, Bmal1
, and Cry1
. These feedback loops, in combination with diverse post-translational mechanisms, result in a rhythmic (about 24 h) expression of clock genes, of their protein product, and of a number of output genes (or clock controlled genes) governing a multitude of physiological functions.
It was initially reported that rhythmic gene expression did not depend on rhythmic binding of CLOCK and BMAL1 to the E-box element of a target gene, that of Per1
in particular 
. More recent reports clearly demonstrate, however, that CLOCK and BMAL1 bind to clock genes, notably to Per1
, and Dbp (D site albumin promoter binding protein)
, in a time-of-day dependent manner in the mouse liver 
. Similar observations of rhythmic chromatin binding were reported also for the core clock transcription factors CLOCK and CYCLE in the fruit fly, where they were shown to contribute to rhythmic gene expression 
. These data imply that, at least for some clock genes, changes in binding of the core clock transcription factors to their genomic sequence play a role in the circadian modulation of their expression.
Work from our group and that of others demonstrate that, in addition to their well established role in generating circadian rhythms, clock genes also play a circadian-independent role in sleep homeostasis 
. First, mutations in some clock gene change the electroencephalographic (EEG) and molecular markers of sleep pressure in mice 
. For instance, Npas2−/−
mice show an attenuated EEG delta power response to sleep deprivation (SD), revealed between 1 and 2 Hz, compared to wild-type littermates 
, whereas this marker of sleep intensity is greatly increased during normal sleep in Cry1,2−/−
double-knockout mice 
. Second, an increase in sleep pressure achieved by SD changes the expression of several clock genes in the forebrain of various inbred mouse strains, notably that of Per1
and Dbp 
. Moreover, although we found that in the mouse some of these sleep/wake-dependent changes, especially those of Per1
, were driven by the corticosterone surge associated with the SD, the increase in Per2
expression and the decrease in Dbp
expression were largely independent of corticosterone 
. This last finding suggests that the SD-induced changes in the expression of these two clock genes are likely caused by modifications in the activity of the core clock transcription factors (i.e., CLOCK, NPAS2, and BMAL1) upon their respective promoter. The fact that the increase in Per2
in the brain is reduced in Npas2−/−
and increased in Cry1,2
double KO mice 
further supports such notion.
We here investigate the potential role of changes in DNA binding activity of core clock transcription factors in the modulation of clock gene expression observed with elevated sleep pressure. Specifically, we hypothesize that SD modifies the binding of clock transcription factors to the E-box and/or E'-box sequences of specific clock genes, especially that of Per2
, in the mouse cerebral cortex. Using chromatin immunoprecipitation (ChIP) of cortical tissue, we first observed that the binding of clock transcription factors to several clock genes depended on time-of-day in the mouse cortex in a way comparable to the time course previously described for liver 
. Importantly, we found that SD alters the binding of clock transcription factors to their target genes. This effect was both transcription factor and target gene specific. Our findings thus reveal that sleep pressure, in addition to internal time-of-day, can modify the DNA-binding properties of core clock proteins in the brain, providing a mechanism by which clock components can sense homeostatic sleep need.