The compaction of genomic DNA into chromatin has profound implications for the regulation of key DNA processes such as transcription. For example, the occupancy of canonical nucleosomes at the TSS of genes prevents access to the transcription machinery. Therefore, considerable attention has been given to the understanding of how these nucleosomes are remodelled, altered or removed to enable transcriptional activation. Significantly, the incorporation of variant histones plays an important role in establishing an active transcriptionally-poised chromatin structure encompassing the TSS. The TSS of active genes transcribed by RNA polymerase II are depleted in stable nucleosomes to create a nucleosome depleted region, which is believed to be necessary for the binding of the transcription machinery. While the mechanism(s) that control the size and the location of the NDR is poorly understood, it is established that the NDR is flanked on both sides by H2A.Z containing-nucleosomes in mammalian cells. We show here that the TSS is not nucleosome free but is occupied by different types of histone variant-containing nucleosomes dependent upon the cellular context. Specifically, in the mouse testes we identified a novel histone variant designated H2A.Lap1 (Lack of acidic patch) that is incorporated into a nucleosome located at the TSS of developmentally regulated genes. Intriguingly, H2A.Z-containing nucleosome at position +1 was missing indicating a novel chromatin organisation for active genes expressed in the mouse testis. Surprisingly in the brain, where H2A.Lap1 is also expressed, it is absent from the TSS with a corresponding gain of the +1 H2A.Z-containing nucleosome. Using mouse trophoblast stem (TS) cells synchronized at different stages of cell cycle, we identified an unstable heterotypic H2A.Z/H2A-containing nucleosome at the TSS in G1 which, strikingly, is lost following DNA replication. Dynamic changes in H2A.Z at the TSS mirror a global expansion of the NDR at S which, unexpectedly, is unrelated to transcriptional activity. We conclude that different strategies utilizing histone variants are used to assemble unstable nucleosomes at the TSS.